Localization of the Interaction Site of Herpes Simplex Virus Glycoprotein D (gD) on the Membrane Fusion Regulator, gH/gL.

A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, and gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL-blocking MAbs were located within flexible regions of the gH N terminus and the gL C terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, "stabilizing" MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV.IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with a receptor to activate the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we have identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibodies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion, which identifies targets for vaccine development.

Surface Plasmon Resonance Reveals Direct Binding of Herpes Simplex Virus Glycoproteins gH/gL to gD and Locates a gH/gL Binding Site on gD.

Herpes simplex virus (HSV) requires fusion between the viral envelope and host membrane. Four glycoproteins, gD, gH/gL, and gB, are essential for this process. To initiate fusion, gD binds its receptor and undergoes a conformational change that hypothetically leads to activation of gH/gL, which in turn triggers the fusion protein gB to undergo rearrangements leading to membrane fusion. Our model predicts that gD must interact with both its receptor and gH/gL to promote fusion. In support of this, we have shown that gD is structurally divided into two "faces": one for the binding receptor and the other for its presumed interaction with gH/gL. However, until now, we have been unable to demonstrate a direct interaction between gD and gH/gL. Here, we used surface plasmon resonance to show that the ectodomain of gH/gL binds directly to the ectodomain of gD when (i) gD is captured by certain anti-gD monoclonal antibodies (MAbs) that are bound to a biosensor chip, (ii) gD is bound to either one of its receptors on a chip, and (iii) gD is covalently bound to the chip surface. To localize the gH/gL binding site on gD, we used multiple anti-gD MAbs from six antigenic communities and determined which ones interfered with this interaction. MAbs from three separate communities block gD-gH/gL binding, and their epitopes encircle a geographical area on gD that we propose comprises the gH/gL binding domain. Together, our results show that gH/gL interacts directly with gD, supporting a role for this step in HSV entry.IMPORTANCE HSV entry is a multistep process that requires the actions of four glycoproteins, gD, gH/gL, and gB. Our current model predicts that gD must interact with both its receptor and gH/gL to promote viral entry. Although we know a great deal about how gD binds its receptors, until now we have been unable to demonstrate a direct interaction between gD and gH/gL. Here, we used a highly sensitive surface plasmon resonance technique to clearly demonstrate that gD and gH/gL interact. Furthermore, using multiple MAbs with defined epitopes, we have delineated a domain on gD that is independent of that used for receptor binding and which likely represents the gH/gL interaction domain. Targeting this interaction to prevent fusion may enhance both therapeutic and vaccine strategies.

Vaccine-induced antibodies to herpes simplex virus glycoprotein D epitopes involved in virus entry and cell-to-cell spread correlate with protection against genital disease in guinea pigs.

Herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) subunit antigen is included in many preclinical candidate vaccines. The rationale for including gD2 is to produce antibodies that block crucial gD2 epitopes involved in virus entry and cell-to-cell spread. HSV-2 gD2 was the only antigen in the Herpevac Trial for Women that protected against HSV-1 genital infection but not HSV-2. In that trial, a correlation was detected between gD2 ELISA titers and protection against HSV-1, supporting the importance of antibodies. A possible explanation for the lack of protection against HSV-2 was that HSV-2 neutralization titers were low, four-fold lower than to HSV-1. Here, we evaluated neutralization titers and epitope-specific antibody responses to crucial gD2 epitopes involved in virus entry and cell-to-cell spread as correlates of immune protection against genital lesions in immunized guinea pigs. We detected a strong correlation between neutralizing antibodies and protection against genital disease. We used a high throughput biosensor competition assay to measure epitope-specific responses to seven crucial gD2 linear and conformational epitopes involved in virus entry and spread. Some animals produced antibodies to most crucial epitopes while others produced antibodies to few. The number of epitopes recognized by guinea pig immune serum correlated with protection against genital lesions. We confirmed the importance of antibodies to each crucial epitope using monoclonal antibody passive transfer that improved survival and reduced genital disease in mice after HSV-2 genital challenge. We re-evaluated our prior study of epitope-specific antibody responses in women in the Herpevac Trial. Humans produced antibodies that blocked significantly fewer crucial gD2 epitopes than guinea pigs, and antibody responses in humans to some linear epitopes were virtually absent. Neutralizing antibody titers and epitope-specific antibody responses are important immune parameters to evaluate in future Phase I/II prophylactic human vaccine trials that contain gD2 antigen.

Characterizing Epitope Binding Regions of Entire Antibody Panels by Combining Experimental and Computational Analysis of Antibody: Antigen Binding Competition.

Vaccines and immunotherapies depend on the ability of antibodies to sensitively and specifically recognize particular antigens and specific epitopes on those antigens. As such, detailed characterization of antibody-antigen binding provides important information to guide development. Due to the time and expense required, high-resolution structural characterization techniques are typically used sparingly and late in a development process. Here, we show that antibody-antigen binding can be characterized early in a process for whole panels of antibodies by combining experimental and computational analyses of competition between monoclonal antibodies for binding to an antigen. Experimental "epitope binning" of monoclonal antibodies uses high-throughput surface plasmon resonance to reveal which antibodies compete, while a new complementary computational analysis that we call "dock binning" evaluates antibody-antigen docking models to identify why and where they might compete, in terms of possible binding sites on the antigen. Experimental and computational characterization of the identified antigenic hotspots then enables the refinement of the competitors and their associated epitope binding regions on the antigen. While not performed at atomic resolution, this approach allows for the group-level identification of functionally related monoclonal antibodies (i.e., communities) and identification of their general binding regions on the antigen. By leveraging extensive epitope characterization data that can be readily generated both experimentally and computationally, researchers can gain broad insights into the basis for antibody-antigen recognition in wide-ranging vaccine and immunotherapy discovery and development programs.

Dynamics of a Viscous Droplet in Return Bends of Microfluidic Channels.

Return bends are frequently encountered in microfluidic systems. In this study, a three-dimensional spectral boundary element method for interfacial dynamics in Stokes flow has been adopted to investigate the dynamics of viscous droplets in rectangular return bends. The droplet trajectory, deformation, and migration velocity are investigated under the influence of various fluid properties and operational conditions, which are depicted by the Capillary number, viscosity ratio, and droplet size, as well as the dimensions of the return bend. While the computational results provide information for the design of return bends in microfluidic systems in general, the computational framework shows potential to guide the design and operation of a droplet-based microfluidic delivery system for cell seeding.

Global sensing of the antigenic structure of herpes simplex virus gD using high-throughput array-based SPR imaging.

While HSV-2 typically causes genital lesions, HSV-1 is increasingly the cause of genital herpes. In addition, neonatal HSV infections are associated with a high rate of mortality and HSV-2 may increase the risk for HIV or Zika infections, reinforcing the need to develop an effective vaccine. In the GSK Herpevac trial, doubly sero-negative women were vaccinated with a truncated form of gD2 [gD2(284t)], then examined for anti-gD serum titers and clinical manifestations of disease. Surprisingly, few vaccinees were protected against genital HSV-2 but 86% were protected from genital HSV-1. These observations suggest that subtle differences in gD structure might influence a protective response. To better understand the antigenic structure of gD and how it impacts a protective response, we previously utilized several key anti-gD monoclonal antibodies (mAbs) to dissect epitopes in vaccinee sera. Several correlations were observed but the methodology limited the number of sera and mAbs that could be tested. Here, we used array-based surface plasmon imaging (SPRi) to simultaneously measure a larger number of protein-protein interactions. We carried out cross-competition or "epitope binning" studies with 39 anti-gD mAbs and four soluble forms of gD, including a form [gD2(285t)] that resembles the Herpevac antigen. The results from these experiments allowed us to organize the mAbs into four epitope communities. Notably, relationships within and between communities differed depending on the form of gD, and off-rate analysis suggested differences in mAb-gD avidity depending on the gD serotype and length. Together, these results show that gD1 and gD2 differ in their structural topography. Consistent with the Herpevac results, several mAbs that bind both gD1 and gD2 neutralize only HSV-1. Thus, this technology provides new insights into the antigenic structure of gD and provides a rationale as to how vaccination with a gD2 subunit may lead to protection from HSV-1 infection.

LPS modification promotes maintenance of Yersinia pestis in fleas.

Yersinia pestis, the causative agent of plague, can be transmitted by fleas by two different mechanisms: by early-phase transmission (EPT), which occurs shortly after flea infection, or by blocked fleas following long-term infection. Efficient flea-borne transmission is predicated upon the ability of Y. pestis to be maintained within the flea. Signature-tagged mutagenesis (STM) was used to identify genes required for Y. pestis maintenance in a genuine plague vector, Xenopsylla cheopis. The STM screen identified seven mutants that displayed markedly reduced fitness in fleas after 4 days, the time during which EPT occurs. Two of the mutants contained insertions in genes encoding glucose 1-phosphate uridylyltransferase (galU) and UDP-4-amino-4-deoxy-l-arabinose-oxoglutarate aminotransferase (arnB), which are involved in the modification of lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N) and resistance to cationic antimicrobial peptides (CAMPs). These Y. pestis mutants were more susceptible to the CAMPs cecropin A and polymyxin B, and produced lipid A lacking Ara4N modifications. Surprisingly, an in-frame deletion of arnB retained modest levels of CAMP resistance and Ara4N modification, indicating the presence of compensatory factors. It was determined that WecE, an aminotransferase involved in biosynthesis of enterobacterial common antigen, plays a novel role in Y. pestis Ara4N modification by partially offsetting the loss of arnB. These results indicated that mechanisms of Ara4N modification of lipid A are more complex than previously thought, and these modifications, as well as several factors yet to be elucidated, play an important role in early survival and transmission of Y. pestis in the flea vector.

An enantiomerically pure formulation of esmolol attenuates hypotension and preserves heart rate control in dogs.

BACKGROUND: Esmolol is marketed as a racemate (RS-esmolol) with hypotension being the most frequently reported adverse event. Previously, it has been shown that the S-enantiomer (S-esmolol) possesses all of the heart rate (HR) control. The authors studied whether S-esmolol alone mitigates hypotension at similar degrees of HR control compared with RS-esmolol. METHODS: The effects of RS- and S-esmolol on blood pressure (BP) were compared at multiple infusion rates producing similar HR control in dogs (N=21). Differences in BP were further interrogated by monitoring global cardiovascular function and included the R-enantiomer (R-esmolol) (N=3). RESULTS: S-esmolol at half the rate (µg kg min) of RS-esmolol provided the same degree of HR control over all infusion rates. RS-esmolol lowered BP by 3, 6, 11, 20, and 38 mmHg at 90, 300, 600, 1,000, and 2,000 µg kg min, compared with 2, 4, 5, 10, and 16 mmHg at 45, 150, 300, 500, and 1,000 µg kg min for S-esmolol. Decreased BP with RS-esmolol was attributed to decreases in left ventricular developed pressure (LVDP) (-34 mmHg), LVdP/dt+max (-702 mmHg/s), and cardiac output (-1 l/min). R-esmolol also decreased BP (-10 mmHg), LVDP (-10 mmHg), LVdP/dt+max (-241 mmHg/s), and cardiac output (to -0.2 l/min). S-esmolol reversed these trends toward pre-esmolol values by increasing BP (+13 mmHg), LVDP (+12 mmHg), LVdP/dt+max (+76 mmHg/s), and cardiac output (+0.4 l/min). CONCLUSIONS: R-enantiomer provided no HR control, but contributed to the hypotension with RS-esmolol, which appears to be due to negative inotropy. Thus, an S-enantiomer formulation of esmolol may provide similar HR control with less hypotension.

A critical comparison of protein microarray fabrication technologies.

Of the diverse analytical tools used in proteomics, protein microarrays possess the greatest potential for providing fundamental information on protein, ligand, analyte, receptor, and antibody affinity-based interactions, binding partners and high-throughput analysis. Microarrays have been used to develop tools for drug screening, disease diagnosis, biochemical pathway mapping, protein-protein interaction analysis, vaccine development, enzyme-substrate profiling, and immuno-profiling. While the promise of the technology is intriguing, it is yet to be realized. Many challenges remain to be addressed to allow these methods to meet technical and research expectations, provide reliable assay answers, and to reliably diversify their capabilities. Critical issues include: (1) inconsistent printed microspot morphologies and uniformities, (2) low signal-to-noise ratios due to factors such as complex surface capture protocols, contamination, and static or no-flow mass transport conditions, (3) inconsistent quantification of captured signal due to spot uniformity issues, (4) non-optimal protocol conditions such as pH, temperature, drying that promote variability in assay kinetics, and lastly (5) poor protein (e.g., antibody) printing, storage, or shelf-life compatibility with common microarray assay fabrication methods, directly related to microarray protocols. Conventional printing approaches, including contact (e.g., quill and solid pin), non-contact (e.g., piezo and inkjet), microfluidics-based, microstamping, lithography, and cell-free protein expression microarrays, have all been used with varying degrees of success with figures of merit often defined arbitrarily without comparisons to standards, or analytical or fiduciary controls. Many microarray performance reports use bench top analyte preparations lacking real-world relevance, akin to "fishing in a barrel", for proof of concept and determinations of figures of merit. This review critiques current protein-based microarray preparation techniques commonly used for analytical and function-based proteomics and their effects on array-based assay performance.

A comparison of the potential for acute cardiopulmonary adverse effects in dogs during continuous veno-venous hemofiltration with accusol 35 solution with and without induced calcium carbonate particles.

BACKGROUND: Baxter received reports of visible precipitate, identified as calcium carbonate, forming during hemofiltration with Accusol 35 solution. AIM: To evaluate the potential for acute cardiopulmonary adverse effects of Accusol 35 containing exaggerated calcium carbonate particles. METHODS: Anesthetized dogs underwent continuous veno-venous hemofiltration (CVVH) with Accusol 35 containing visible and subvisible particles (≥10 µm) 36 times higher than the maximum concentration specified in the European Pharmacopoeia (P-Accusol), or Accusol 35 conforming to specification (Accusol). Select cardiovascular and blood gas parameters were evaluated during CVVH. Lung tissue samples were collected following CVVH. RESULTS: No differences were observed in cardiovascular and blood gas parameters or lung histology between P-Accusol and Accusol. CONCLUSION: Accusol 35 containing visible and subvisible particles (≥10 µm) 36 times higher than the maximum concentration specified in the European Pharmacopoeia resulted in no acute cardiopulmonary adverse effects compared with Accusol 35 containing no visible particles and subvisible particles within European Pharmacopoeia specification.

Hell's Itch: A Unique Reaction to UV Exposure.

We present a survey-based exploration of Hell's Itch, a severe dermatologic reaction often mistaken for sunburn, that reveals distinct symptoms including intense pain, unrelenting itching, paresthesia, and even suicidal ideation, differentiating it from a typical sunburn.

The Correlation of Sleep Disturbance and Location of Glioma Tumors: A Narrative Review.

Sleep disturbance can occur when sleep centers of the brain, regions that are responsible for coordinating and generating healthy amounts of sleep, are disrupted by glioma growth or surgical resection. Several disorders cause disruptions to the average duration, quality, or patterns of sleep, resulting in sleep disturbance. It is unknown whether specific sleep disorders can be reliably correlated with glioma growth, but there are sufficient numbers of case reports to suggest that a connection is possible. In this manuscript, these case reports and retrospective chart reviews are considered in the context of the current primary literature on sleep disturbance and glioma diagnosis to identify a new and useful connection which warrants further systematic and scientific examination in preclinical animal models. Confirmation of the relationship between disruption of the sleep centers in the brain and glioma location could have significant implications for diagnostics, treatment, monitoring of metastasis/recurrence, and end-of-life considerations.

Cationic PAMAM dendrimers disrupt key platelet functions.

Poly(amidoamine) (PAMAM) dendrimers have been proposed for a variety of biomedical applications and are increasingly studied as model nanomaterials for such use. The dendritic structure features both modular synthetic control of molecular size and shape and presentation of multiple equivalent terminal groups. These properties make PAMAM dendrimers highly functionalizable, versatile single-molecule nanoparticles with a high degree of consistency and low polydispersity. Recent nanotoxicological studies showed that intravenous administration of amine-terminated PAMAM dendrimers to mice was lethal, causing a disseminated intravascular coagulation-like condition. To elucidate the mechanisms underlying this coagulopathy, in vitro assessments of platelet functions in contact with PAMAM dendrimers were undertaken. This study demonstrates that cationic G7 PAMAM dendrimers activate platelets and dramatically alter their morphology. These changes to platelet morphology and activation state substantially altered platelet function, including increased aggregation and adherence to surfaces. Surprisingly, dendrimer exposure also attenuated platelet-dependent thrombin generation, indicating that not all platelet functions remained intact. These findings provide additional insight into PAMAM dendrimer effects on blood components and underscore the necessity for further research on the effects and mechanisms of PAMAM-specific and general nanoparticle toxicity in blood.

Moving beyond Titers.

Vaccination to prevent and even eliminate disease is amongst the greatest achievements of modern medicine. Opportunities remain in vaccine development to improve protection across the whole population. A next step in vaccine development is the detailed molecular characterization of individual humoral immune responses against a pathogen, especially the rapidly evolving pathogens. New technologies such as sequencing the immune repertoire in response to disease, immunogenomics/vaccinomics, particularly the individual HLA variants, and high-throughput epitope characterization offer new insights into disease protection. Here, we highlight the emerging technologies that could be used to identify variation within the human population, facilitate vaccine discovery, improve vaccine safety and efficacy, and identify mechanisms of generating immunological memory. In today's vaccine-hesitant climate, these techniques used individually or especially together have the potential to improve vaccine effectiveness and safety and thus vaccine uptake rates. We highlight the importance of using these techniques in combination to understand the humoral immune response as a whole after vaccination to move beyond neutralizing titers as the standard for immunogenicity and vaccine efficacy, especially in clinical trials.

Antibody Printing Technologies.

Antibody microarrays are routinely employed in the lab and in the clinic for studying protein expression, protein-protein, and protein-drug interactions. The microarray format reduces the size scale at which biological and biochemical interactions occur, leading to large reductions in reagent consumption and handling times while increasing overall experimental throughput. Specifically, antibody microarrays, as a platform, offer a number of different advantages over traditional techniques in the areas of drug discovery and diagnostics. While a number of different techniques and approaches have been developed for creating micro and nanoscale antibody arrays, issues relating to sensitivity, cost, and reproducibility persist. The aim of this review is to highlight current state-of the-art techniques and approaches for creating antibody arrays by providing latest accounts of the field while discussing potential future directions.

Epitope characterization of anti-drug antibodies-a tool for discovery and health: an overview of the necessity of early epitope characterization to avoid anti-drug antibodies and promote patient health.

Introduction: The market for monoclonal antibody (mAb) therapies is growing rapidly as the pharmaceutical industry expands its development across a broad spectrum of diseases. Unfortunately, as shown in the recent failure of bococizumab by Pfizer, these treatments often stimulate the formation of problematic anti-drug antibodies (ADAs). ADAs can cause side effects and limit efficacy for many patients. To increase efficacy and decrease safety concerns from ADAs, immunogenicity characterization is needed early in the drug development process. Here, we present emerging techniques that hold promise to improve ADA assays and their potential applications to pharmaceutical development and personalized medicine.Areas covered: This manuscript outlines the importance of epitope characterization to better understand immunogenicity and describes a strategy for using this information in treating patients taking mAb therapies.Expert opinion: We propose using high-information assays to characterize epitopes to help mAb therapy engineering and potentially improve individual patient outcomes. To understand this, we will discuss three different aspects of ADAs: (1) the problem of ADAs and what is currently being done about them, (2) the current state of epitope characterization and how it is being utilized, and (3) how early epitope characterization can advance drug discovery and improve outcomes for patients taking mAb therapies.

The next frontier of oncotherapy: accomplishing clinical translation of oncolytic bacteria through genetic engineering.

The development of a 'smart' drug capable of distinguishing tumor from host cells has been sought for centuries, but the microenvironment of solid tumors continues to confound therapeutics. Solid tumors present several challenges for current oncotherapeutics, including aberrant vascularization, hypoxia, necrosis, abnormally high pH and local immune suppression. While traditional chemotherapeutics are limited by such an environment, oncolytic microbes are drawn to it - having an innate ability to selectively infect, colonize and eradicate solid tumors. Development of an oncolytic species would represent a shift in the cancer therapeutic paradigm, with ramifications reaching from the medical into the socio-economic. Modern genetic engineering techniques could be implemented to customize 'Frankenstein' bacteria with advantageous characteristics from several species.

Lay abstract Side effects of chemotherapeutics are thought to often be a reflection of our inability to target these toxic substances to only cancer cells; hence, scientists have spent centuries searching for alternative treatments that would confine their actions to tumor cells, sparing healthy tissue. Unfortunately, the dense nature of tumor tissue along with altered blood vessels, that lead to diminished tumor tissue oxygenation, altered tissue pH and cellular metabolic inactivity or even cell death have proven challenging. Importantly, these barriers have contributed to local and even sometimes systemic suppression of the patient's immune system that can allow the tumor to grow and progress unchecked. While most non-cancer cells are inhibited by the local tumor environment, certain microbes, including some bacteria and viruses, are drawn to it, possessing a natural ability to selectively infect, colonize and eradicate solid tumors. These microbes may also restore the patient's immune balance. However, use of these microbes is not without its own problems; nevertheless, modern genetic engineering techniques could be implemented to develop customized, safe, effective bacteria with advantageous characteristics. The development and clinical translation of cancer-fighting bacteria would represent a shift in cancer therapeutics and would have ramifications that reach beyond medical efficacy into the realm of socioeconomics. This review seeks to marry the current field of oncolytic bacteria with the expanding field of modern bacterial genetic engineering techniques in prospect of such a therapeutic.

Potential Therapeutic Targets for Combination Antibody Therapy against Pseudomonas aeruginosa Infections.

Despite advances in antimicrobial therapy and even the advent of some effective vaccines, Pseudomonas aeruginosa (P. aeruginosa) remains a significant cause of infectious disease, primarily due to antibiotic resistance. Although P. aeruginosa is commonly treatable with readily available therapeutics, these therapies are not always efficacious, particularly for certain classes of patients (e.g., cystic fibrosis (CF)) and for drug-resistant strains. Multi-drug resistant P. aeruginosa infections are listed on both the CDC's and WHO's list of serious worldwide threats. This increasing emergence of drug resistance and prevalence of P. aeruginosa highlights the need to identify new therapeutic strategies. Combinations of monoclonal antibodies against different targets and epitopes have demonstrated synergistic efficacy with each other as well as in combination with antimicrobial agents typically used to treat these infections. Such a strategy has reduced the ability of infectious agents to develop resistance. This manuscript details the development of potential therapeutic targets for polyclonal antibody therapies to combat the emergence of multidrug-resistant P. aeruginosa infections. In particular, potential drug targets for combinational immunotherapy against P. aeruginosa are identified to combat current and future drug resistance.

Efficient screening of high-signal and low-background antibody pairs in the bio-bar code assay using prion protein as the target.

The bio-bar code assay is an assay for ultrasensitive detection of proteins. The main technical hurdle in bio-bar code assay development is achieving a dose-dependent, reproducible signal with low background. We report on a magnetic bead ELISA screening mechanism for characterizing antibody pairs that are effective for use in the bio-bar code assay. The normal isoform of prion protein was utilized as the target protein as dozens of antibodies have been developed against it. The development of an ultrasensitive assay for the detection of the various isoforms of PrP has the potential to enable significant advances in the diagnosis and understanding of transmissible spongiform encephalopathies, including transmission mechanisms, disease pathology, and potential therapeutics. With prion protein as the target, the magnetic bead ELISA identified pairs with high background and low signal in the bio-bar code assay. The magnetic bead ELISA was effective as a screening mechanism because it reduced assay time and cost and allowed for understanding of pair characteristics such as development times and signal-to-noise ratios.

Using hydrodynamic focusing to predictably alter the diameter of synthetic silk fibers.

Spiders and silkworms provide a model of superior processing for multifunctional and highly versatile high-performance fibers. Mimicking the spider's complex control system for chemical and mechanical gradients has remained an ongoing obstacle for synthetic silk production. In this study, the use of hydrodynamic fluid focusing within a 3D printed biomimetic spinning system to recapitulate the biological spinneret is explored and shown to produce predictable, small diameter fibers. Mirroring in silico fluid flow simulations using a hydrodynamic microfluidic spinning technique, we have developed a model correlating spinning rates, solution viscosity and fiber diameter outputs that will significantly advance the field of synthetic silk fiber production. The use of hydrodynamic focusing to produce controlled output fiber diameter simulates the natural silk spinning process and continues to build upon a 3D printed biomimetic spinning platform.