investigating-receptor-ligand-systems-cellulosome-with-afm-based

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

The purpose of this study was to examine ovarian cancer cells for the expression of decorin, a proteoglycan component of the cell matrix that can inhibit cancer cell growth.

The expression of decorin in human ovarian tumors.

The purpose is to determine dose-limiting toxicity, pharmacokinetics,pharmacodynamics, and immunobiology after i.p. injections of recombinant human IL-12 (rhIL-12).

Phase I study of intraperitoneal recombinant human interleukin 12 in patients with Müllerian carcinoma, gastrointestinal primary malignancies, and mesothelioma.

Although membrane channel proteins are important to drug discovery and hold great promise as engineered nanopore sensing elements, their widespread application to these areas has been limited by difficulties in fabricating planar lipid-bilayer membranes. We present a method for forming these sub-5-nm-thick free-standing structures based on a self-assembly process driven by solvent extraction in a microfluidic channel. This facile automatable process forms high-quality membranes able to host channel proteins measurable at single-molecule conductance resolution.

Automated formation of lipid-bilayer membranes in a microfluidic device.

Exotic slugs have become serious pests of canola, at establishment, in Southern Australian high-rainfall cropping zones. Slugs were monitored using relatively inexpensive 300 mm x 300 mm terracotta tiles acting as refuges. An investigation was made of the effects of the time of application of chelated iron baits on the slug species Deroceras reticulatum Müller and Lehmannia nyctelia Bourguignat. Baits reduced the number of surface-active slug species. A single application at sowing provided greater efficacy than one application before sowing, and efficacy was comparable with that of two applications. Canola seedling densities showed a negative response to D. reticulatum numbers; the presence of even one individual per refuge trap reduced seedling numbers below optimum densities. Thistles and other vegetation were associated with increased numbers of slugs. European guidelines for slug monitoring and damage appear to be at least partly applicable to Australian conditions.

Slug control in Australian canola: monitoring, molluscicidal baits and economic thresholds.

Parthenogenetic species are assumed to represent evolutionary dead ends, yet parthenogenesis is common in some groups of invertebrates particularly in those found in relatively constant environments. This suggests that parthenogenetic reproduction might be common in pest invertebrates from uniform agricultural environments. Based on the evaluations of two databases from North America and Italy, we found that parthenogenetic species comprised 45 per cent (North America) or 48 per cent (Italy) of pest species derived from genera where parthenogenesis occurred, compared with an overall incidence of 10 per cent or 16 per cent in these genera. In establishing these patterns, we included only genera containing at least some member species that reproduced by parthenogenesis. The high incidence of parthenogenesis in pest species is spread across different families and several insect orders. Parthenogenetic reproduction may be favoured in agricultural environments when particular clones have a high fitness across multiple generations. Increasing the complexity and variability of agricultural environments represents one way of potentially controlling parthenogenetic pest species.

A high incidence of parthenogenesis in agricultural pests.

In the absence of applied forces, the transport of molecules and particulate reagents across laminar flowstreams in microfluidic devices is dominated by the diffusivities of the transported species. While the differential diffusional properties between smaller and larger diagnostic targets and reagents have been exploited for bioseparation and assay applications, there are limitations to methods that depend on these intrinsic size differences. Here a new strategy is described for exploiting the sharply reversible change in size and magnetophoretic mobility of "smart" magnetic nanoparticles (mNPs) to perform bioseparation and target isolation under continuous flow processing conditions. The isolated 5 nm mNPs do not exhibit significant magnetophoretic velocities, but do exhibit high magnetophoretic velocities when aggregated by the action of a pH-responsive polymer coating. A simple external magnet is used to magnetophorese the aggregated mNPs that have captured a diagnostic target from a lower pH laminar flowstream (pH 7.3) to a second higher pH flowstream (pH 8.4) that induces rapid mNP disaggregation. In this second dis-aggregated state and flowstream, the mNPs continue to flow past the magnet rather than being immobilized at the channel surface near the magnet. This stimuli-responsive reagent system has been shown to transfer 81% of a model protein target from an input flowstream to a second flowstream in a continuous flow H-filter device.

Dynamic bioprocessing and microfluidic transport control with smart magnetic nanoparticles in laminar-flow devices.

We report a new strategy for synthesizing temperature-responsive gamma-Fe(2)O(3)-core/Au-shell nanoparticles (Au-mNPs) from diblock copolymer micelles. The amphiphilic diblock copolymer chains were synthesized using reversible addition-fragmentation chain-transfer (RAFT) with a thermally responsive "smart" poly(N-isopropylacrylamide) (pNIPAAm) block and an amine-containing poly(N,N-dimethylaminoethylacrylamide) (DMAEAm) block that acted as a reducing agent during gold shell formation. The Au-mNPs reversibly aggregated upon heating the solution above the transition temperature of pNIPAAm, resulting in a red-shifted localized surface plasmon resonance.

"Smart" diblock copolymers as templates for magnetic-core gold-shell nanoparticle synthesis.

We describe the synthesis and characterization of high-performance, superparamagnetic, iron oxide nanoparticle-based, heavy metal sorbents, which demonstrate excellent affinity for the separation of heavy metals in contaminated water systems (i.e., spiked Columbia River water). The magnetic nanoparticle sorbents were prepared from an easy-to-synthesize iron oxide precursor, followed by a simple, one-step ligand exchange reaction to introduce an affinity ligand to the nanoparticle surface that is specific to a heavy metal or class of heavy metal contaminants. The engineered magnetic nanoparticle sorbents have inherently high active surface areas, allowing for increased binding capacities. To demonstrate the performance of the nanoparticle sorbents, river water was spiked with specific metals and exposed to low concentrations of the functionalized nanoparticles. In almost all cases, the nanoparticles were found to be superior to commercially available sorbent materials as well as the unfunctionalized iron oxide nanoparticles.

High-performance, superparamagnetic, nanoparticle-based heavy metal sorbents for removal of contaminants from natural waters.

A method for patterning narrow lines of biomolecules onto nitrocellulose membranes using laboratory syringe pumps is described. One syringe pump is used to drive the biomolecule solution through a needle, while another modified syringe pump acts as a one-dimensional translation stage, moving the needle across the membrane much like a pen. This method consumes very small volumes of reagents, and is a viable option for laboratory-scale fabrication and prototyping of point-of-care rapid diagnostic test strips.

Laboratory-scale protein striping system for patterning biomolecules onto paper-based immunochromatographic test strips.

Communities of arthropods providing ecosystem services (e.g., pest control, pollination, and soil nutrient cycling) to agricultural production systems are influenced by pesticide inputs, yet the impact of pesticide applications on nontarget organisms is normally evaluated through standardized sets of laboratory tests involving individual pesticides applied to a few representative species. By combining season-long pesticide applications of various insecticides and fungicides into a metric based on the International Organization for Biological and Integrated Control (IOBC) toxicity ratings, we evaluate season-long pesticide impacts on communities of indigenous and exotic arthropods across 61 vineyards assessed for an entire growing season. The composition of arthropod communities, identified mostly at the family level, but in some cases at the species level, was altered depending on season-long pesticide use. Numbers of mostly indigenous parasitoids, predatory mites, and coccinellids in the canopy, as well as carabid/tenebrionid beetles and some spider families on the ground, were decreased at higher cumulative pesticide metric scores. In contrast, numbers of one invasive millipede species (Ommatoiulus moreletti Lucas, Julida: Julidae) increased under higher cumulative pesticide metric scores. These changing community patterns were detected despite the absence of broad-spectrum insecticide applications in the vineyards. Pesticide effects were mostly due to indoxacarb and sulphur, applied as a fungicide. The reduction of beneficial arthropods and increase in an invasive herbivorous millipede under high cumulative pesticide metric scores highlights the need to manage nontarget season-long pesticide impacts in vineyards. A cumulative pesticide metric, based on IOBC toxicity ratings, provides a way of assessing overall toxicity effects, giving managers a means to estimate and consider potential negative season-long pesticide impacts on ecosystem services provided through arthropod communities.

Identifying signature of chemical applications on indigenous and invasive nontarget arthropod communities in vineyards.

A new diagnostic system for the enrichment and detection of protein biomarkers from human plasma is presented. Gold nanoparticles (AuNPs) were surface-modified with a diblock copolymer synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. The diblock copolymer contained a thermally responsive poly(N-isopropylacrylamide) (pNIPAAm) block, a cationic amine-containing block, and a semi-telechelic PEG₂-biotin end group. When a mixed suspension of 23 nm pNIPAAm-modified AuNPs was heated with pNIPAAm-coated 10 nm iron oxide magnetic nanoparticles (mNPs) in human plasma, the thermally responsive pNIPAAm directed the formation of mixed AuNP/mNP aggregates that could be separated efficiently with a magnet. Model studies showed that this mixed nanoparticle system could efficiently purify and strongly enrich the model biomarker protein streptavidin in spiked human plasma. A 10 ng/mL streptavidin sample was mixed with the biotinylated pNIPAAm-modified AuNPs and magnetically separated in the mixed nanoparticle system with pNIPAAm mNPs. The aggregates were concentrated into a 50-fold smaller fluid volume at room temperature where the gold nanoparticle reagent redissolved with the streptavidin target still bound. The concentrated gold-labeled streptavidin could be subsequently analyzed directly using lateral flow immunochromatography. This rapid capture and enrichment module thus utilizes the mixed stimuli-responsive nanoparticle system to achieve concentration of a gold-labeled biomarker that can be directly analyzed using lateral flow or other rapid diagnostic strategies.

Mixed stimuli-responsive magnetic and gold nanoparticle system for rapid purification, enrichment, and detection of biomarkers.

There is a need for simple yet robust biomarker and antigen purification and enrichment strategies that are compatible with current rapid diagnostic modalities. Here, a stimuli-responsive nanoparticle system is presented for multiplexed magneto-enrichment and non-instrumented lateral flow strip detection of model antigens from spiked pooled plasma. The integrated reagent system allows purification and enrichment of the gold-labeled biomarker half-sandwich that can be applied directly to lateral flow test strips. A linear diblock copolymer with a thermally responsive poly(N-isopropylacrylamide) (pNIPAm) segment and a gold-binding block composed of NIPAm-co-N,N-dimethylaminoethylacrylamide was prepared by reversible addition-fragmentation chain transfer polymerization. The diblock copolymer was used to functionalize gold nanoparticles (AuNPs), with subsequent bioconjugation to yield thermally responsive pNIPAm-AuNPs that were co-decorated with streptavidin. These AuNPs efficiently complexed biotinylated capture antibody reagents that were bound to picomolar quantities of pan-aldolase and Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in spiked pooled plasma samples. The gold-labeled biomarker half-sandwich was then purified and enriched using 10 nm thermally responsive magnetic nanoparticles that were similarly decorated with pNIPAm. When a thermal stimulus was applied in conjunction with a magnetic field, coaggregation of the AuNP half-sandwiches with the pNIPAm-coated iron oxide nanoparticles created large aggregates that were efficiently magnetophoresed and separated from bulk serum. The purified biomarkers from a spiked pooled plasma sample could be concentrated 50-fold into a small volume and applied directly to a commercial multiplexed lateral flow strip to dramatically improve the signal-to-noise ratio and test sensitivity.

Multiplexed enrichment and detection of malarial biomarkers using a stimuli-responsive iron oxide and gold nanoparticle reagent system.

Besteht ein Bedarf f&uuml;r einfache, aber robuste Biomarker und Antigen-Reinigung und Bereicherung Strategien, die mit aktuellen rasche diagnostische Modalit&auml;ten kompatibel sind. Hier ist ein Reize-responsive Nanopartikel-System f&uuml;r Multiplex Magneto-Anreicherung und nicht instrumentiert lateral-Flow Streifen Detektion von Modell-Antigene von Spike gepoolte Plasma pr&auml;sentiert. Das integrierte Reagenz-System erm&ouml;glicht Reinigung und Bereicherung der Gold-Label Biomarker halb-Sandwich, direkt die lateral-Flow Teststreifen angewendet werden kann. Eine lineare Diblock-Copolymer mit einem thermisch reagierende Coil (pNIPAm)-Segment und einem Gold-Bindung-Block bestehend aus NIPAm-co-N, N-Dimethylaminoethylacrylamide wurde durch reversible Addition-Fragmentierung Kette Transfer Polymerisation vorbereitet. Die Diblock Copolymer wurde zur Goldene-Nanopartikeln (AuNPs), Stra&szlig;enschlachten mit nachfolgenden Bioconjugation, thermisch reagierende pNIPAm-AuNPs zu erbringen, die co-decorated mit Streptavidin waren. Diese AuNPs effizient komplexiert biotinylierten Erfassung Antik&ouml;rper Reagenzien, die in pikolomaren Mengen von Pan-Aldolase und Plasmodium Falciparum Histidin-reiche Protein 2 (PfHRP2) gebunden wurden Pfennigabsatz gepoolte Plasmaproben. Der Gold-Label Biomarker halb-Sandwich wurde dann gereinigt und angereichert mit 10 nm thermisch reagierende magnetischer Nanopartikel, die in &auml;hnlicher Weise dekoriert wurden mit pNIPAm. Wann ein thermischer Reiz in Verbindung mit einem Magnetfeld, Coaggregation der AuNP halb-Sandwiches mit pNIPAm-beschichteten Eisenoxid Nanopartikel erstellt gro&szlig;e Aggregate angewendet wurde, die von Massen Serum getrennt und wurden effizient Magnetophoresed. Die gereinigten Biomarker aus einem Spike gepoolte Plasma-Beispiel k&ouml;nnte konzentrierte 50-fache, in einem kleinen Volumen und angewendet, die direkt an einen kommerziellen Multiplex-lateral-Flow-Streifen, die Signal-Rausch-Verh&auml;ltnis und Test-Empfindlichkeit drastisch zu verbessern.

Multiplex Bereicherung und Erkennung von Malariagebiete Biomarkern, die mit einem Reize-responsive Eisenoxid und gold-Nanopartikel-Reagenz-System.

Adhesion of environmentally responsive polymers to biocompatible surfaces is an important issue that has been explored in several nanobiotechnology applications. Here, we prepared multi-responsive statistical copolymers of two oligo(ethylene glycol) methyl ether methacrylate macromonomers with differing ethylene glycol side chain lengths using RAFT polymerization. The lower critical solution temperature of the copolymers was characterized using visible light extinction, and the chemical composition and molecular weight were measured using NMR spectroscopy and size-exclusion chromatography, respectively. The characterization results demonstrated that the transition temperature could be controlled by varying the macromonomer feed ratios, and the molecular weight could be controlled by varying the amount of the RAFT chain transfer agent in the polymerization feed. Using AFM single-molecule force spectroscopy, we measured the adhesion characteristics of single copolymer molecules to a gold surface. We found that dehydration and collapse of the copolymer in a high ionic strength buffer resulted in dramatically reduced bridging length distributions that maintained their single-molecule bimodal character. In the collapsed state, the polymer exhibited a lower absolute desorption force while cooperativity effects were found to increase the desorption force per chain for multi-chain interactions. Our results confirmed that the polymer in a collapsed conformation exhibited a dramatically reduced volume occupancy above the gold surface. These results demonstrate at the single-molecule level how solvent-induced collapse of an environmentally responsive copolymer modulates surface adhesion forces and bridging length distributions in a controllable way.

Single-molecule adhesion of a stimuli-responsive oligo(ethylene glycol) copolymer to gold.

Cellulose-degrading enzyme systems are of significant interest from both a scientific and technological perspective due to the diversity of cellulase families, their unique assembly and substrate binding mechanisms, and their potential applications in several key industrial sectors, notably cellulose hydrolysis for second-generation biofuel production. Particularly fascinating are cellulosomes, the multimodular extracellular complexes produced by numerous anaerobic bacteria. Using single-molecule force spectroscopy, we analyzed the mechanical stability of the intermolecular interfaces between the cohesin and the dockerin modules responsible for self-assembly of the cellulosomal components into the multienzyme complex. The observed cohesin-dockerin rupture forces (>120 pN) are among the highest reported for a receptor-ligand system to date. Using an atomic force microscope protocol that quantified single-molecule binding activity, we observed force-induced dissociation of calcium ions from the duplicated loop-helix F-hand motif located within the dockerin module, which in the presence of EDTA resulted in loss of affinity to the cohesin partner. A cohesin amino acid mutation (D39A) that eliminated hydrogen bonding with the dockerin's critically conserved serine residues reduced the observed rupture forces. Consequently, no calcium loss occurred and dockerin activity was maintained throughout multiple forced dissociation events. These results offer insights at the single-molecule level into the stability and folding of an exquisite class of high-affinity protein-protein interactions that dictate fabrication and architecture of cellulose-degrading molecular machines.

Article	Year
The expression of decorin in human ovarian tumors. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 12060613	2002
Phase I study of intraperitoneal recombinant human interleukin 12 in patients with Müllerian carcinoma, gastrointestinal primary malignancies, and mesothelioma. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 12473577	2002
Automated formation of lipid-bilayer membranes in a microfluidic device. Nano letters\| PubMed ID: 16968008	2006
Slug control in Australian canola: monitoring, molluscicidal baits and economic thresholds. Pest management science\| PubMed ID: 17592623	2007
A high incidence of parthenogenesis in agricultural pests. Proceedings. Biological sciences / The Royal Society\| PubMed ID: 18647717	2008
Dynamic bioprocessing and microfluidic transport control with smart magnetic nanoparticles in laminar-flow devices. Lab on a chip\| PubMed ID: 19568666	2009
"Smart" diblock copolymers as templates for magnetic-core gold-shell nanoparticle synthesis. Nano letters\| PubMed ID: 20017498	2010
High-performance, superparamagnetic, nanoparticle-based heavy metal sorbents for removal of contaminants from natural waters. ChemSusChem\| PubMed ID: 20468024	2010
Laboratory-scale protein striping system for patterning biomolecules onto paper-based immunochromatographic test strips. Lab on a chip\| PubMed ID: 20607151	2010
Identifying signature of chemical applications on indigenous and invasive nontarget arthropod communities in vineyards. Ecological applications : a publication of the Ecological Society of America\| PubMed ID: 20945768	2010
Mixed stimuli-responsive magnetic and gold nanoparticle system for rapid purification, enrichment, and detection of biomarkers. Bioconjugate chemistry\| PubMed ID: 21070026	2010
Multiplexed enrichment and detection of malarial biomarkers using a stimuli-responsive iron oxide and gold nanoparticle reagent system. ACS nano\| PubMed ID: 22804625	2012
Single-molecule adhesion of a stimuli-responsive oligo(ethylene glycol) copolymer to gold. ACS nano\| PubMed ID: 23113802	2012
Single-molecule dissection of the high-affinity cohesin-dockerin complex. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23188794	2012

Michael A. Nash

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