Fully Aqueous Self-Assembly of a Gold-Nanoparticle-Based Pathogen Sensor.

Surface plasmon resonance (SPR) is a very sensitive measure of biomolecular interactions but is generally too expensive for routine analysis of clinical samples. Here we demonstrate the simplified formation of virus-detecting gold nanoparticle (AuNP) assemblies on glass using only aqueous buffers at room temperature. The AuNP assembled on silanized glass and displayed a distinctive absorbance peak due to the localized SPR (LSPR) response of the AuNPs. Next, assembly of a protein engineering scaffold was followed using LSPR and a sensitive neutron reflectometry approach, which measured the formation and structure of the biological layer on the spherical AuNP. Finally, the assembly and function of an artificial flu sensor layer consisting of an in vitro-selected single-chain antibody (scFv)-membrane protein fusion was followed using the LSPR response of AuNPs within glass capillaries. In vitro selection avoids the need for separate animal-derived antibodies and allows for the rapid production of low-cost sensor proteins. This work demonstrates a simple approach to forming oriented arrays of protein sensors on nanostructured surfaces that uses (i) an easily assembled AuNP silane layer, (ii) self-assembly of an oriented protein layer on AuNPs, and (iii) simple highly specific artificial receptor proteins.

Opposite changes in the expression of clathrin and caveolin-1 in normal and cancerous human prostate tissue: putative clathrin-mediated recycling of EGFR.

Endocytosis, an important macromolecule uptake process in cells, is known to be dysregulated in cancer. Clathrin and caveolin-1 proteins play a major role in receptor-mediated endocytosis. We have used a quantitative, unbiased and semi-automated method to measure in situ protein expression of clathrin and caveolin-1 in cancerous and paired normal (cancer adjacent, non-cancerous) human prostate tissue. There was a significant (p < 0.0001) increase in the expression of clathrin in prostate cancer samples (N = 29, n = 91) compared to normal tissue (N = 29, n = 67) (N = number of patients, n = number of cores in tissue arrays). Conversely, there was a significant (p < 0.0001) decrease in expression of caveolin-1 in prostate cancer tissue compared to normal prostate tissue. The opposite change in expression of the two proteins was highly correlated to increasing cancer aggressiveness. There was also a concurrent increase in the expression of epidermal growth factor receptor (EGFR), a key receptor in carcinogenesis, with clathrin in prostate cancer tissue, indicating recycling of EGFR through clathrin-mediated endocytosis (CME). These results indicate that in prostate cancer, caveolin-1-mediated endocytosis (CavME) may be acting as a brake and increase in CME may facilitate tumorigenicity and aggressiveness of prostate cancer through recycling of EGFR. Changes in the expression of these proteins can also potentially be used as a biomarker for prostate cancer to aid in diagnosis and prognosis and clinical decision-making.

Modular Protein Engineering Approach to the Functionalization of Gold Nanoparticles for Use in Clinical Diagnostics.

Functional protein-gold nanoparticle (AuNP) conjugates have a wide variety of applications including biosensing and drug delivery. Correct protein orientation, which is important to maintain functionality on the nanoparticle surface, can be difficult to achieve in practice, and dedicated protein scaffolds have been used on planar gold surfaces to drive the self-assembly of oriented protein arrays. Here we use the transmembrane domain of Escherichia coli outer membrane protein A (OmpATM) to create protein-AuNP conjugates. The addition of a single cysteine residue into a periplasmic loop, to create cysOmpATM, drives oriented assembly and increased equilibrium binding. As the protein surface concentration increases, the sulfur-gold bond in cysOmpATM creates a more densely populated AuNP surface than the poorly organized wtOmpATM layer. The functionalization of AuNP improved both their stability and homogeneity. This was further exploited using multidomain protein chimeras, based on cysOmpATM, which were shown to form ordered protein arrays with their functional domains displayed away from the AuNP surface. A fusion with protein G was shown to specifically bind antibodies via their Fc region. Next, an in vitro selected single chain antibody (scFv)-cysOmpATM fusion protein, bound to AuNP, detected influenza A nucleoprotein, a widely used antigen in diagnostic assays. Finally, using the same scFv-cysOmpATM-AuNP conjugates, a prototype lateral flow assay for influenza demonstrated the utility of fully recombinant self-assembling sensor layers. By simultaneously removing the need for both animal antibodies and a separate immobilization procedure, this technology could greatly simplify the development of a range of in vitro diagnostics.

Detecting Selective Protein Binding Inside Plasmonic Nanopores: Toward a Mimic of the Nuclear Pore Complex.

Biosensors based on plasmonic nanostructures offer label-free and real-time monitoring of biomolecular interactions. However, so do many other surface sensitive techniques with equal or better resolution in terms of surface coverage. Yet, plasmonic nanostructures offer unique possibilities to study effects associated with nanoscale geometry. In this work we use plasmonic nanopores with double gold films and detect binding of proteins inside them. By thiol and trietoxysilane chemistry, receptors are selectively positioned on the silicon nitride interior walls. Larger (~150 nm) nanopores are used detect binding of averaged sized proteins (~60 kg/mol) with high signal to noise (>100). Further, we fabricate pores that approach the size of the nuclear pore complex (diameter down to 50 nm) and graft disordered phenylalanine-glycine nucleoporin domains to the walls, followed by titration of karyopherinß1 transport receptors. The interactions are shown to occur with similar affinity as determined by conventional surface plasmon resonance on planar surfaces. Our work illustrates another unique application of plasmonic nanostructures, namely the possibility to mimic the geometry of a biological nanomachine with integrated optical sensing capabilities.

Investigating recycled water use as a diffuse source of per- and polyfluoroalkyl substances (PFASs) to groundwater in Melbourne, Australia.

The purpose of this study was to investigate the contribution of per- and polyfluoroalkyl substances (PFASs) to groundwater at a location where recycled water from a wastewater treatment plant (WWTP) is used to irrigate crops. Groundwater from Werribee South, located west of Melbourne, Australia, was sampled over two campaigns in 2017 and 2018, extracted using solid phase extraction (SPE) and analysed with liquid chromatography-tandem mass spectrometry (LC-MS/MS-QQQ). PFASs were detected in 100% of the groundwater samples. The sum total of twenty PFAS compounds (∑20PFASs) for all sites in the study ranged from <0.03 to 74 ng/L (n = 28) and the highest levels of which were observed in the centre of the irrigation district. Perfluorooctanesulfonic acid (PFOS) was the most detected compound overall (96%) with a mean concentration of 11 ng/L (<0.03-34 ng/L), followed by perfluorobutanesulfonic acid (PFBS; 86%, 4.4 ng/L), perfluorooctanoic acid (PFOA; 82%, 2.2 ng/L) and perfluorobutanoic acid (PFBA; 77%, 6.1 ng/L). Concentrations of PFASs found in this study are greater than background levels of PFASs detected in groundwater and are in the range of concentrations typically detected in wastewater effluent. This study presents evidence that the use of recycled water can be a source of PFAS contamination to groundwater.

Repertoire of HLA-DR1-restricted CD4 T-cell responses to capsular Caf1 antigen of Yersinia pestis in human leukocyte antigen transgenic mice.

Yersinia pestis is the causative agent of plague, a rapidly fatal infectious disease that has not been eradicated worldwide. The capsular Caf1 protein of Y. pestis is a protective antigen under development as a recombinant vaccine. However, little is known about the specificity of human T-cell responses for Caf1. We characterized CD4 T-cell epitopes of Caf1 in "humanized" HLA-DR1 transgenic mice lacking endogenous major histocompatibility complex class II molecules. Mice were immunized with Caf1 or each of a complete set of overlapping synthetic peptides, and CD4 T-cell immunity was measured with respect to proliferative and gamma interferon T-cell responses and recognition by a panel of T-cell hybridomas, as well as direct determination of binding affinities of Caf1 peptides to purified HLA-DR molecules. Although a number of DR1-restricted epitopes were identified following Caf1 immunization, the response was biased toward a single immunodominant epitope near the C terminus of Caf1. In addition, potential promiscuous epitopes, including the immunodominant epitope, were identified by their ability to bind multiple common HLA alleles, with implications for the generation of multivalent vaccines against plague for use in humans.

Synergistic collagenase expression and cartilage collagenolysis are phosphatidylinositol 3-kinase/Akt signaling-dependent.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has emerged as a major regulator of cellular functions and has been implicated in several pathologies involving remodeling of extracellular matrix (ECM). The end stage of inflammatory joint diseases is characterized by excessive ECM catabolism, and in this study we assess the role of PI3K signaling in the induction of collagenolytic matrix metalloproteinases (MMPs) in human chondrocytes. We used the most potent cytokine stimulus reported to promote cartilage ECM catabolism, namely interleukin-1 (IL-1) in combination with oncostatin M (OSM). Both OSM and IL-6 (in the presence of its soluble receptor), but not IL-1 nor leukemia inhibitory factor, induced Akt phosphorylation in human chondrocytes. Inhibition of PI3K signaling using LY294002 blocked IL-1+OSM-mediated Akt phosphorylation, induction of MMP-1 and MMP-13, and cartilage collagenolysis. To further explore the role of downstream substrates within the PI3K pathway, complementary use of small molecule inhibitors and specific small interfering RNAs demonstrated that the PI3K subunit p110alpha and Akt1 were required for MMP-1 mRNA induction. MMP-13 induction was also reduced by loss of function of these molecules and by a lack of p110delta, 3-phosphoinositide-dependent kinase-1 or Akt3. We therefore propose that the activities of specific elements of the PI3K signaling pathway, including Akt, are necessary for the synergistic induction of MMP-1 and MMP-13 and the cartilage breakdown stimulated by IL-1+OSM. Our data provide new insight into the mechanism of synergy between IL-1 and OSM and highlight new therapeutic targets for inflammatory joint diseases that aim to repress the expression of collagenases.

The multiple signals assessed by female satin bowerbirds: could they be used to narrow down females' choices of mates?

Female choice based on multiple male traits has been documented in many species but the functions of such multiple traits are still under debate. The satin bowerbird has a polygynous mating system in which males attract females to bowers for mating; females choose mates based on multiple aspects of males and their bowers. In this paper, we demonstrate that females use some cues to decide which males to examine closely and other cues to decide which males to mate with. Female visitation rates to bowers were significantly related to male size and the males' 'solitary' display rates, and, to a lesser extent, to the numbers of bower decorations. After controlling for female visitation rates, it was found that a male's mating success was significantly related to his size and the rate at which he 'painted' his bower with saliva and chewed up plant material.