Functionalized magnetic nanoparticles (mNPs) have shown promise in biosensing and other biomedical applications. Here we use functionalized mNPs to develop a highly sensitive, versatile sensing strategy required in practical biological assays and potentially in vivo analysis. We demonstrate a new sensing scheme based on magnetic spectroscopy of nanoparticle Brownian motion (MSB) to quantitatively detect molecular target. MSB uses the harmonics of oscillating mNPs as a metric for the freedom of rotational motion, thus reflecting the bound state of the mNP. The harmonics can be detected in vivo from nanogram quantities of iron within 5 seconds. Using a streptavidin-biotin binding system, we show that the detection limit of the current MSB technique is lower than 150 pM (0.075 picomole), which is much more sensitive than previously reported techniques based on mNP detection. Using mNPs conjugated with two anti-thrombin DNA aptamers, we show that thrombin can be detected with high sensitivity (4 nM or 2 picomole). A DNA-DNA interaction was also investigated. The results demonstrated that sequence selective DNA detection can be achieved with 100 pM (2 nM) sensitivity. The results of using MSB to sense these interactions, verified that the MSB based sensing technique can achieve rapid measurement (within 10 sec), and is suitable for detecting and quantifying a wide range of biomarkers or analytes. It has the potential to be applied in variety of biomedical applications or diagnostic analyses.

We describe an ultra-high-throughput screening platform enabling discovery and/or engineering of natural product antibiotics. The methodology involves creation of hydrogel-in-oil emulsions in which recombinant microorganisms are co-emulsified with bacterial pathogens; antibiotic activity is assayed by use of a fluorescent viability dye. We have successfully utilized both bulk emulsification and microfluidic technology for the generation of hydrogel microdroplets that are size-compatible with conventional flow cytometry. Hydrogel droplets are ~25 pL in volume, and can be synthesized and sorted at rates exceeding 3,000 drops/s. Using this technique, we have achieved screening throughputs exceeding 5 million clones/day. Proof-of-concept experiments demonstrate efficient selection of antibiotic-secreting yeast from a vast excess of negative controls. In addition, we have successfully used this technique to screen a metagenomic library for secreted antibiotics that kill the human pathogen Staphylococcus aureus. Our results establish the practical utility of the screening platform, and we anticipate that the accessible nature of our methods will enable others seeking to identify and engineer the next generation of antibacterial biomolecules.

More than 2 decades of study support the hypothesis that alginate lyases are promising therapeutic candidates for treating mucoid Pseudomonas aeruginosa infections. In particular, the enzymes' ability to degrade alginate, a key component of mucoid biofilm matrix, has been the presumed mechanism by which they disrupt biofilms and enhance antibiotic efficacy. The systematic studies reported here show that, in an in vitro model, alginate lyase dispersion of P. aeruginosa biofilms and enzyme synergy with tobramycin are completely decoupled from catalytic activity. In fact, equivalent antibiofilm effects can be achieved with bovine serum albumin or simple amino acids. These results provide new insights into potential mechanisms of alginate lyase therapeutic activity, and they should motivate a careful reexamination of the fundamental assumptions underlying interest in enzymatic biofilm dispersion. P remature mortality among cystic fibrosis (CF) patients is typically associated with chronic airway infection by the opportunistic pathogen Pseudomonas aeruginosa (1). Upon colonization of the airway, P. aeruginosa causes a range of clinical complications, including stimulation of a hyperinflammatory response, obstruction of the pulmonary tract, and progressive loss of lung function (1-3). The bacterium's persistence within the CF lung is thought to result in large part from a biofilm mode of growth (2, 4), which can subvert both the host immune response and antibacterial therapies (5). In contrast to environmental niches, in the CF lung, P. aeruginosa generally transitions to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate (6). This copolymer of (1,4)-linked ␤-D-mannuronic acid and ␣-L-guluronic acid alters biofilm architecture and function (7) and thereby compounds P. aeruginosa's persistence in the chronically inflamed airway (5). Indeed, alginate is one of the most extensively studied P. aeruginosa virulence factors (8).Given alginate's contribution to mucoid biofilm structure, its function in bacterial virulence, and its role in the persistent nature of lung infections, it has long been considered an attractive target for interventional therapies (6). In particular, biocatalytic degradation of mucoid P. aeruginosa biofilms using alginate lyase enzymes (EC 4.2.2.3) has been the subject of more than 20 years of research. Alginate lyase treatment has been shown to reduce viscosity in cultures of clinical isolates and in CF sputum (9, 10); it strips biofilms from abiotic surfaces (11, 12), it enhances phagocytosis and killing of P. aeruginosa by human immune cells (13)(14)(15), and it improves the efficacy of various antipseudomonal antibiotics (14,(16)(17)(18)(19). In aggregate, these reports make a rather convincing case for the use of inhaled alginate lyases for treating chronic P. aeruginosa infections of the CF lung, although no clinical trials have been conducted to date.In an effort to further bolster the rationale for therapeutic alginate lyases, we set out to conduct systemati...

Pseudomonas aeruginosa colonises the upper airway of cystic fibrosis (CF) patients, providing a reservoir of host-adapted genotypes that subsequently establish chronic lung infection. We previously experimentally-evolved P. aeruginosa in a murine model of respiratory tract infection and observed early-acquired mutations in pmrB, encoding the sensor kinase of a two-component system that promoted establishment and persistence of infection. Here, using proteomics, we show downregulation of proteins involved in LPS biosynthesis, antimicrobial resistance and phenazine production in pmrB mutants, and upregulation of proteins involved in adherence, lysozyme resistance and inhibition of the chloride ion channel CFTR, relative to wild-type strain LESB65. Accordingly, pmrB mutants are susceptible to antibiotic treatment but show enhanced adherence to airway epithelial cells, resistance to lysozyme treatment, and downregulate host CFTR expression. We propose that P. aeruginosa pmrB mutations in CF patients are subject to an evolutionary trade-off, leading to enhanced colonisation potential, CFTR inhibition, and resistance to host defences, but also to increased susceptibility to antibiotics.

Therapeutic proteins continue to yield revolutionary new treatments for a growing spectrum of human disease, but the development of these powerful drugs requires solving a unique set of challenges. For instance, it is increasingly apparent that mitigating potential anti-therapeutic immune responses, driven by molecular recognition of a therapeutic protein's peptide fragments, may be best accomplished early in the drug development process. One may eliminate immunogenic peptide fragments by mutating the cognate amino acid sequences, but deimmunizing mutations are constrained by the need for a folded, stable, and functional protein structure. These two concerns may be competing, as the mutations that are best at reducing immunogenicity often involve amino acids that are substantially different physicochemically. We develop a novel approach, called EpiSweep, that simultaneously optimizes both concerns. Our algorithm identifies sets of mutations making such Pareto optimal trade-offs between structure and immunogenicity, embodied by a molecular mechanics energy function and a T-cell epitope predictor, respectively. EpiSweep integrates structure-based protein design, sequence-based protein deimmunization, and algorithms for finding the Pareto frontier of a design space. While structure-based protein design is NP-hard, we employ integer programming techniques that are efficient in practice. Furthermore, EpiSweep only invokes the optimizer once per identified Pareto optimal design. We show that EpiSweep designs of regions of the therapeutics erythropoietin and staphylokinase are predicted to outperform previous experimental efforts. We also demonstrate EpiSweep's capacity for deimmunization of the entire proteins, case analyses involving dozens of predicted epitopes, and tens of thousands of unique side-chain interactions. Ultimately, Epi-Sweep is a powerful protein design tool that guides the protein engineer toward the most promising immunotolerant biotherapeutic candidates.