Yersinia pseudotuberculosis growth arrest during type-III secretion system expression is associated with altered ribosomal protein expression and decreased gentamicin susceptibility.

It has been long appreciated that expression of the Yersinia type-III secretion system (T3SS) in culture is associated with growth arrest. Here we sought to understand whether this impacts expression of ribosomal protein genes, which were among the most highly abundant transcripts in exponential phase Yersinia pseudotuberculosis based on RNA-seq analysis. To visualize changes in ribosomal protein expression, we generated a fluorescent transcriptional reporter with the promoter upstream of rpsJ/S10 fused to a destabilized gfp variant. We confirmed reporter expression significantly increases in exponential phase and decreases as cells transition to stationary phase. We then utilized a mouse model of systemic Y. pseudotuberculosis infection to compare T3SS and S10 reporter expression during clustered bacterial growth in the spleen, and found that cells expressing high levels of the T3SS had decreased S10 levels, while cells with lower T3SS expression retained higher S10 expression. In bacteriological media, growth inhibition with T3SS induction and a reduction in S10 expression were observed in subsets of cells, while cells with high expression of both T3SS and S10 were also observed. Loss of T3SS genes resulted in rescued growth and heightened S10 expression. To understand if clustered growth impacted bacterial gene expression, we utilized droplet-based microfluidics to encapsulate bacteria in spherical agarose droplets, and also observed growth inhibition with high expression of T3SS and reduced S10 levels that better mirrored phenotypes observed in the mouse spleen. Finally, we show that T3SS expression is sufficient to promote tolerance to the ribosome-targeting antibiotic, gentamicin. Collectively, these data indicate that the growth arrest associated with T3SS induction leads to decreased expression of ribosomal protein genes, and this results in reduced antibiotic susceptibility.

SBA-15 with Crystalline Walls Produced via Thermal Treatment with the Alkali and Alkali Earth Metal Ions.

Crystalline walled SBA-15 with large pore size were prepared using alkali and alkali earth metal ions (Na+, Li+, K+ and Ca2+). For this work, the ratios of alkali metal ions (Si/metal ion) ranged from 2.1 to 80, while the temperatures tested ranged from 500 to 700 °C. The SBA-15 prepared with Si/Na+ ratios ranging from 2.1 to 40 at 700 °C exhibited both cristobalite and quartz SiO2 structures in pore walls. When the Na+ amount increased (i.e., Si/Na increased from 80 to 40), the pore size was increased remarkably but the surface area and pore volume of the metal ion-based SBA-15 were decreased. When the SBA-15 prepared with Li+, K+ and Ca2+ ions (Si/metal ion = 40) was thermally treated at 700 °C, the crystalline SiO2 of quartz structure with large pore diameter (i.e., 802.5 Å) was observed for Ca+2 ion-based SBA-15, while no crystalline SiO2 structures were observed in pore walls for both the K+ and Li+ ions treated SBA-15. The crystalline SiO2 structures may be formed by the rearrangement of silica matrix when alkali or alkali earth metal ions are inserted into silica matrix at elevated temperature.

Reprogramming Virus Coat Protein Carboxylate Interactions for the Patterned Assembly of Hierarchical Nanorods.

The self-assembly system of the rod-shaped tobacco mosaic virus (TMV) has been studied extensively for nanoscale applications. TMV coat protein assembly is modulated by intersubunit carboxylate groups whose electrostatic repulsion limits the assembly of virus rods without incorporating genomic RNA. To engineer assembly control into this system, we reprogrammed intersubunit carboxylate interactions to produce self-assembling coat proteins in the absence of RNA and in response to unique pH and ionic environmental conditions. Specifically, engineering a charge attraction at the intersubunit E50-D77 carboxylate group through a D77K substitution stabilized the coat proteins assembly into virus-like rods. In contrast, the reciprocal E50K modification alone did not confer virus-like rod assembly. However, a combination of R46G/E50K/E97G substitutions enabled virus-like rod assembly. Interestingly, the D77K substitution displays a unique pH-dependent assembly-disassembly profile, while the R46G/E50K/E97G substitutions confer a novel salt concentration dependency for assembly control. In addition, these unique environmentally controlled coat proteins allow for the directed assembly and disassembly of chimeric virus-like rods both in solution and on substrate-attached seed rods. Combined, these findings provide a controllable means to assemble functionally discrete virus-like rods for use in nanotechnology applications.

Integrated System for Bacterial Detection and Biofilm Treatment on Indwelling Urinary Catheters.

GOAL: This work introduces an integrated system incorporated seamlessly with a commercial Foley urinary catheter for bacterial growth sensing and biofilm treatment. METHODS: The system is comprised of flexible, interdigitated electrodes incorporated with a urinary catheter via a 3D-printed insert for impedance sensing and bioelectric effect-based treatment. Each of the functions were wirelessly controlled using a custom application that provides a user-friendly interface for communicating with a custom PCB via Bluetooth to facilitate implementation in practice. RESULTS: The integrated catheter system maintains the primary functions of indwelling catheters - urine drainage, balloon inflation - while being capable of detecting the growth of Escherichia coli, with an average decrease in impedance of 13.0% after 24 hours, tested in a newly-developed simulated bladder environment. Furthermore, the system enables bioelectric effect-based biofilm reduction, which is performed by applying a low-intensity electric field that increases the susceptibility of biofilm bacteria to antimicrobials, ultimately reducing the required antibiotic dosage. CONCLUSION: Overall, this modified catheter system represents a significant step forward for catheter-associated urinary tract infection (CAUTI) management using device-based approaches, integrating flexible electrodes with an actual Foley catheter along with the control electronics and mobile application. SIGNIFICANCE: CAUTIs, exacerbated by the emergence of antibiotic-resistant pathogens, represent a significant challenge as one of the most prevalent healthcare-acquired infections. These infections are driven by the colonization of indwelling catheters by bacterial biofilms.

Light-Activated Polymer-Coated Mesoporous Silica with Azobenzene Moiety for the Controlled Delivery of Guest Molecules.

In this study, we have synthesized a light-activated polymer-coated mesoporous silica nanovalve with poly(trans-4-methacryloyloxyazobenzene-co-methylmethacrylic acid-co-vinyltrimethoxysilane) (PMMV) (MSNs/PMMV) for the controlled delivery of guest molecules. The hydrophobicity of azobenzene varies depending on the structure of each isomer. Typically, trans isomers are hydrophobic due to the aromatic ring, but they become more hydrophilic when they are changed to the cis confirmation. Using this concept, we introduced PMMV as a light active nanovalve on the mesoporous silica. To optimize the coating of the light-activated polymer on mesoporous silica, we investigated the conformational change of PMMV in solutions at various pHs. PMMV has a dot-like morphology in acidic solutions under pH 4, but sheet-like morphology in solutions at pH over 4. We investigated the nanovalve behavior of MSNs/PMMV by introducing propidium iodide (PI) as guest molecules. Time-resolved fluorescence spectroscopy showed the excellent light activity of PMMV for open pores. The new PMMV-coated mesoporous silica could be applied in the smart nanovalve system for the controlled delivery of various guest molecules.

Ingestible Sensors and Sensing Systems for Minimally Invasive Diagnosis and Monitoring: The Next Frontier in Minimally Invasive Screening.

Ingestible electronic systems that are capable of embedded sensing, particularly within the gastrointestinal (GI) tract and its accessory organs, have the potential to screen for diseases that are difficult if not impossible to detect at an early stage using other means. Furthermore, these devices have the potential to (1) reduce labor and facility costs for a variety of procedures, (2) promote research for discovering new biomarker targets for associated pathologies, (3) promote the development of autonomous or semiautonomous diagnostic aids for consumers, and (4) provide a foundation for epithelially targeted therapeutic interventions. These technological advances have the potential to make disease surveillance and treatment far more effective for a variety of conditions, allowing patients to lead longer and more productive lives. This review will examine the conventional techniques, as well as ingestible sensors and sensing systems that are currently under development for use in disease screening and diagnosis for GI disorders. Design considerations, fabrication, and applications will be discussed.

Microsystems for biofilm characterization and sensing - A review.

Biofilms are the primary cause of clinical bacterial infections and are impervious to typical amounts of antibiotics, necessitating very high doses for elimination. Therefore, it is imperative to have suitable methods for characterization to develop novel methods of treatment that can complement or replace existing approaches using significantly lower doses of antibiotics. This review presents some of the current developments in microsystems for characterization and sensing of bacterial biofilms. Initially, we review current standards for studying biofilms that are based on invasive and destructive end-point biofilm characterization. Additionally, biofilm formation and growth is extremely sensitive to various growth and environmental parameters that cause large variability in biofilms between repeated experiments, making it very difficult to compare experimental repeats and characterize the temporal characteristics of these organisms. To address these challenges, recent developments in the field have moved toward systems and miniature devices that can aid in the non-invasive characterization of bacterial biofilms. Our review focuses on several types of microsystems for biofilm evaluation including optical, electrochemical, and mechanical systems. This review will show how these devices can lead to better understanding of the physiology and function of these communities of bacteria, which can eventually lead to the development of novel treatments that do not rely on high-dosage antibiotics.

Tobacco Mosaic Virus as a Versatile Platform for Molecular Assembly and Device Fabrication.

Viruses are unique biological agents that infect living host cells through molecular delivery of a genomic cargo. Over the past two decades advancements in genetic engineering and bioconjugation technologies have allowed the unprecedented use of these "unfriendly" biological molecules, as nanoscopic platforms for the advancement of an array of nanotechnology applications. This mini-review focuses on providing a brief summary of key demonstrations leveraging the versatile characteristics of Tobacco mosaic virus (TMV) for molecular assembly and bio-device integration. A comprehensive discussion of genetic and chemical modification strategies along with potential limiting factors that impact the assembly of these macromolecules is presented to provide useful insights for adapting TMV as a potentially universal platform toward developing advanced nanomaterials. Additional discussions on biofabrication techniques developed in parallel to enable immobilization, alignment, and patterning of TMV-based functional particles on solid surfaces will highlight technological innovations that can be widely adapted for creating nanoscopic device components using these engineered biomacromolecules. Further exploitation in the design of molecular specificity and assembly mechanisms and the development of highly controllable and scalable TMV-device integration strategies will expand the library of nanoscale engineering tools that can be used for the further development of virus-based nanotechnology platforms.

Localized Three-Dimensional Functionalization of Bionanoreceptors on High-Density Micropillar Arrays via Electrowetting.

In this work, we introduce an electrowetting-assisted 3-D biofabrication process allowing both complete and localized functionalization of bionanoreceptors onto densely arranged 3-D microstructures. The integration of biomaterials with 3-D microdevice components offers exciting opportunities for communities developing miniature bioelectronics with enhanced performance and advanced modes of operation. However, most biological materials are stable only in properly conditioned aqueous solutions, thus the water-repellent properties exhibited by densely arranged micro/nanostructures (widely known as the Cassie-Baxter state) represent a significant challenge to biomaterial integration. Here, we first investigate such potential limitations using cysteine-modified tobacco mosaic virus (TMV1cys) as a model bionanoreceptor and a set of Au-coated Si-micropillar arrays (µPAs) of varying densities. Furthermore, we introduce a novel biofabrication system adopting electrowetting principles for the controlled localization of TMV1cys bionanoreptors on densely arranged µPAs. Contact angle analysis and SEM characterizations provide clear evidence to indicate structural hydrophobicity as a key limiting factor for 3-D biofunctionalization and for electrowetting as an effective method to overcome this limitation. The successful 3-D biofabrication is confirmed using SEM and fluorescence microscopy that show spatially controlled and uniform assemblies of TMV1cys on µPAs. The increased density of TMV1cys per device footprint produces a 7-fold increase in fluorescence intensity attributed to the µPAs when compared to similar assemblies on planar substrates. Combined, this work demonstrates the potential of electrowetting as a unique enabling solution for the controlled and efficient biofabrication of 3-D-patterned micro/nanodomains.

Biofabrication of Tobacco mosaic virus-nanoscaffolded supercapacitors via temporal capillary microfluidics.

This paper reports the implementation of temporal capillary microfluidic patterns and biological nanoscaffolds in autonomous microfabrication of nanostructured symmetric electrochemical supercapacitors. A photoresist layer was first patterned on the substrate, forming a capillary microfluidics layer with two separated interdigitated microchannels. Tobacco mosaic virus (TMV) macromolecules suspended in solution are autonomously delivered into the microfluidics, and form a dense bio-nanoscaffolds layer within an hour. This TMV layer is utilized in the electroless plating and thermal oxidation for creating nanostructured NiO supercapacitor. The galvanostatic charge/discharge cycle showed a 3.6-fold increase in areal capacitance for the nanostructured electrode compared to planar structures. The rapid creation of nanostructure-textured microdevices with only simple photolithography and bionanostructure self-assembly can completely eliminate the needs for sophisticated synthesis or deposition processes. This method will contribute to rapid prototyping of wide range of nano-/micro-devices with enhanced performance.

Novel fluorinated polysilsesquioxane hollow spheres: synthesis and application in drug release.

Fluorinated polysilsesquioxane (FPSQ) hollow spheres with a large empty interior were synthesized in an aqueous medium by using (trifluoropropyl)trimethoxysilane as the sole precursor. The drug release applications of these spheres were demonstrated, and the materials have great potential as fluorinated drug release carriers.