Functionalizing Yeast Lipid Droplets as Versatile Biomaterials.

Lipid droplets (LD) are dynamic cellular organelles of ≈1 µm diameter in yeast where a neutral lipid core is surrounded by a phospholipid monolayer and attendant proteins. Beyond the storage of lipids, opportunities for LD engineering remain underdeveloped but they show excellent potential as new biomaterials. In this research, LD from yeast Saccharomyces cerevisiae is engineered to display mCherry fluorescent protein, Halotag ligand binding protein, plasma membrane binding v-SNARE protein, and carbonic anhydrase enzyme via linkage to oleosin, an LD anchoring protein. Each protein-oleosin fusion is coded via a single gene construct. The expressed fusion proteins are specifically displayed on LD and their functions can be assessed within cells by fluorescence confocal microscopy, TEM, and as isolated materials via AFM, flow cytometry, spectrophotometry, and by enzyme activity assay. LD isolated from the cell are shown to be robust and stabilize proteins anchored into them. These engineered LD function as reporters, bind specific ligands, guide LD and their attendant proteins into union with the plasma membrane, and catalyze reactions. Here, engineered LD functions are extended well beyond traditional lipid storage toward new material applications aided by a versatile oleosin platform anchored into LD and displaying linked proteins.

Engineer RNA-Protein Nanowires as Light-Responsive Biomaterials.

RNA molecules have emerged as increasingly attractive biomaterials with important applications such as RNA interference (RNAi) for cancer treatment and mRNA vaccines against infectious diseases. However, it remains challenging to engineer RNA biomaterials with sophisticated functions such as non-covalent light-switching ability. Herein, light-responsive RNA-protein nanowires are engineered to have such functions. It first demonstrates that the high affinity of RNA aptamer enables the formation of long RNA-protein nanowires through designing a dimeric RNA aptamer and an engineered green fluorescence protein (GFP) that contains two TAT-derived peptides at N- and C- termini. GFP is then replaced with an optogenetic protein pair system, LOV2 (light-oxygen-voltage) protein and its binding partner ZDK (Z subunit of protein A), to confer blue light-controlled photo-switching ability. The light-responsive nanowires are long (>500 nm) in the dark, but small (20-30 nm) when exposed to light. Importantly, the co-assembly of this RNA-protein hybrid biomaterial does not rely on the photochemistry commonly used for light-responsive biomaterials, such as bond formation, cleavage, and isomerization, and is thus reversible. These RNA-protein structures can serve as a new class of light-controlled biocompatible frameworks for incorporating versatile elements such as RNA, DNA, and enzymes.

Rapid production of multimeric RNA aptamers stabilized by a designed pseudo-circular structure in E. coli.

RNA aptamers bind specifically and selectively to various macromolecules, cell surfaces, and viruses and find broad applications as biosensors, diagnostics, and in therapeutic treatments and drug delivery. Currently, RNA aptamer production is via in vitro methods. Herein, a new E. coli-based approach has been demonstrated for the rapid production of multimeric RNA aptamer transcripts that are protected from degradation by burying the 5' and 3' ends of the transcript in a designed double-stranded spacer. Multimeric and fluorescent RNA aptamers were produced stably in vivo and readily isolated from RNase III-deficient cells, and their full functionalities were shown by binding assays and fluorescence measurements. This approach shows promise as a rapid and scalable bioprocess for the production of RNA aptamers at low cost.

Engineering Self-Assembled Endolysin Nanoparticles against Antibiotic-Resistant Bacteria.

Antibiotic resistance represents a serious global health concern and has stimulated the development of antimicrobial nanomaterials to combat resistant bacteria. Protein-based nanoparticles combining characteristics of both proteins and nanoparticles offer advantages including high biocompatibility, attractive biodegradability, enhanced bioavailability and functional versatility. They have played an increasing role as promising candidates for broad applications ranging from biocatalysts and drug delivery to vaccine development to cancer therapeutics. However, their application as antibacterial biomaterials to address challenging antibiotic-resistance problems has not been explicitly pursued. Herein, we describe engineering protein-only nanoparticles against resistant Gram-positive bacteria. A self-assembling peptide (P114) enables the assembly of a phage lytic enzyme (P128) into nanoparticles in response to pH reduction. Compared to native P128 and monomeric P114-P128, P128 nanoparticles (P128NANO) demonstrated a stronger bactericidal ability with high potency at lower concentrations (2-3-fold lower), particularly for methicillin-resistant Staphylococcus aureus strains. In addition, P128NANO showed an enhanced thermal (up to 65 °C) and storage stability and elicited extensive damages to bacterial cell walls. These remarkable antibacterial abilities are likely due to the P128NANO nanostructure, mediating multivalent interactions with bacterial cell walls at increased local concentrations of endolysin. The engineered endolysin nanoparticles offer a promising antimicrobial alternative to conventional antibiotics.

A Lotus shaped acoustofluidic mixer: High throughput homogenisation of liquids in 2 ms using hydrodynamically coupled resonators.

This paper presents an acoustically actuated microfluidic mixer that uses an array of hydrodynamically coupled resonators to rapidly homogenise liquid solutions and synthesise nanoparticles. The system relies on 8 identical oscillating cantilevers that are equally spaced on the perimeter of a circular well, through which the liquid solutions are introduced. When an oscillatory electrical signal is applied to a piezoelectric transducer attached to the device, the cantilevers start resonating. Due to the close proximity between the cantilevers, their circular arrangement and the liquid medium in which they are immersed, the vibration of each cantilever affects the response of its neighbours. The streaming fields and shearing rates resulting from the oscillating structures were characterised. It was shown that the system can be used to effectively mix fluids at flow rates up to 1400 µl.min-1 in time scales as low as 2 ms. The rapid mixing time is especially advantageous for nanoparticle synthesis, which is demonstrated by synthesising Poly lactide-co-glycolic acid (PLGA) nanoparticles with 52.2 nm size and PDI of 0.44.

High throughput acoustic microfluidic mixer controls self-assembly of protein nanoparticles with tuneable sizes.

HYPOTHESIS: Protein nanoparticles have attracted increased interest due to their broad applications ranging from drug delivery and vaccines to biocatalysts and biosensors. The morphology and the size of the nanoparticles play a crucial role in determining their suitability for different applications. Yet, effectively controlling the size of the nanoparticles is still a significant challenge in their manufacture. The hypothesis of this paper is that the assembly conditions and size of protein particles can be tuned via a mechanical route by simply modifying the mixing time and strength, while keeping the chemical parameters constant. EXPERIMENTAL: We use an acoustically actuated, high throughput, ultrafast, microfluidic mixer for the assembly of protein particles with tuneable sizes. The performance of the acoustic micro-mixer is characterized via Laser Doppler Vibrometry and image processing. The assembly of protein nanoparticles is monitored by dynamic light scattering (DLS) and transmission electron microscopy (TEM). FINDINGS: By changing actuation parameters, the turbulence and mixing in the microchannel can be precisely varied to control the initiation of protein particle assembly while the solution conditions of assembly (pH and ionic strength) are kept constant. Importantly, mixing times as low as 6 ms can be achieved for triggering protein assembly in the microfluidic channel. In comparison to the conventional batch process of assembly, the acoustic microfluidic mixer approach produces smaller particles with a more uniform size distribution, promising a new way to manufacture protein particles with controllable quality.

Open-Closed Structure of Light-Responsive Protein LOV2 Regulates Its Molecular Interaction with a Binding Partner.

Optogenetic approaches have broad applications, including regulating cell signaling and gene expression. Photoresponsive protein LOV2 and its binding partner ZDK represent an important protein caging/uncaging optogenetic system. Herein, we combine time-resolved small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) to reveal different structural states of LOV2 and the light-controlled mechanism of interaction between LOV2 and ZDK. In response to blue light within a time frame of ca. 70 s, LOV2 has a significantly higher value of radius of gyration Rg (29.6 ± 0.3 vs 26.4 ± 0.4 Å) than its dark state, suggesting unwinding of the C-terminal Jα-helix into an open structure. Atomic force microscopy was used to characterize molecular interactions of LOV2 in open and closed states with ZDK at a single-molecule level. The closed state of LOV2 enables strong binding with ZDK, characterized by a 60-fold lower dissociation rate and a ∼1.5-times higher activation energy barrier than for its open state. In combination, these data support a light-switching mechanism that is modulated by the proximity of multiple binding sites of LOV2 for ZDK.

High rates of CTX-M group-1 extended-spectrum ß-lactamases producing Escherichia coli from pets and their owners in Faisalabad, Pakistan.

PURPOSE: Pet animals have been considered a potential carrier of clinically important multi-drug-resistant Escherichia coli. However, little is known about the role of pets as reservoirs of extended-spectrum ß-lactamase (ESBL) producing E. coli in Pakistan. This study was designed to determine the prevalence and genetic relatedness of ESBL-producing multidrug-resistant E. coli in pets, their owners, and veterinary professionals. METHODS: A total of 105 fecal samples were collected from dogs, cats, their owners, and veterinary professionals from veterinary clinics. Isolates of ESBL-producing E. coli were subjected to antimicrobial susceptibility testing. The presence of bla CTX-M genes and CTX-M groups I and II in multidrug-resistant E. coli was detected using PCR. Clonal diversity was checked using BOX-PCR. RESULTS: Of the 105 fecal samples screened, 73 (69.5%) were found to contain ESBL-producing E. coli. The percentage of ESBL-producing E. coli isolates in dogs and dog owners was found to be 81.8% (18/22) and 59% (13/22), respectively. In cats, this percentage was 73.9% (17/23) and in cat owners, 56.5% (13/23). Furthermore, 80% (12/15) of E. coli isolates in veterinary professionals were ESBL producers. Of these 73 ESBL-producing E. coli isolates, 23 isolates exhibited a multidrug-resistant phenotype. The most prevalent multidrug-resistant pattern (17.4%) identified was resistant to ampicillin, cefotaxime, ciprofloxacin, and nitrofurantoin. In the multidrug-resistant E. coli, bla CTX-M was identified as the most common ESBL-producing genotype (19/23), with bla CTX-M-1 dominating in all 19 isolates. Furthermore, BOX-PCR analysis exhibited genetically diverse clonal groups among isolates of the CTX-M-1 group. CONCLUSION: Our results provide important baseline information on the potential burden of multidrug-resistant E. coli among companion animals in Pakistan. Further studies are needed to understand the drivers of antimicrobial resistance at human-animal-environmental intersections.