Cinchona Alkaloid Polymers Demonstrate Highly Efficient Gene Delivery Dependent on Stereochemistry, Methoxy Substitution, and Length.

Nucleic acid delivery with cationic polymers is a promising alternative to expensive viral-based methods; however, it often suffers from a lower performance. Herein, we present a highly efficient delivery system based on cinchona alkaloid natural products copolymerized with 2-hydroxyethyl acrylate. Cinchona alkaloids are an attractive monomer class for gene delivery applications, given their ability to bind to DNA via both electrostatics and intercalation. To uncover the structure-activity profile of the system, four structurally similar cinchona alkaloids were incorporated into polymers: quinine, quinidine, cinchonine, and cinchonidine. These polymers differed in the chain length, the presence or absence of a pendant methoxy group, and stereochemistry, all of which were found to alter gene delivery performance and the ways in which the polymers overcome biological barriers to transfection. Longer polymers that contained the methoxy-bearing cinchona alkaloids (i.e., quinine and quinidine) were found to have the best performance. These polymers exhibited the tightest DNA binding, largest and most abundant DNA-polymer complexes, and best endosomal escape thanks to their increased buffering capacity and closest nuclear proximity of the payload. Overall, this work highlights the remarkable efficiency of polymer systems that incorporate cinchona alkaloid natural products while demonstrating the profound impact that small structural changes can have on overcoming biological hurdles associated with gene delivery.

Vibrio parahaemolyticus and Vibrio vulnificus in vitro biofilm dispersal from microplastics influenced by simulated human environment.

Growing concerns exist regarding human ingestion of contaminated seafood that contains Vibrio biofilms on microplastics (MPs). One of the mechanisms enhancing biofilm related infections in humans is due to biofilm dispersion, a process that triggers release of bacteria from biofilms into the surrounding environment, such as the gastrointestinal tract of human hosts. Dispersal of cells from biofilms can occur in response to environmental conditions such as sudden changes in temperature, pH and nutrient conditions, as the bacteria leave the biofilm to find a more stable environment to colonize. This study evaluated how brief exposures to nutrient starvation, elevated temperature, different pH levels and simulated human media affect Vibrio parahaemolyticus and Vibrio vulnificus biofilm dispersal and processes on and from low-density polyethylene (LDPE), polypropylene (PP), and polystyrene (PS) MPs. Both species were able to adequately disperse from all types of plastics under most exposure conditions. V. parahaemolyticus was able to tolerate and survive the low pH that resembles the gastric environment compared to V. vulnificus. pH had a significantly (p ≤ 0.05) positive effect on overall V. parahaemolyticus biofilm biomass in microplates and cell colonization from PP and PS. pH also had a positive effect on V. vulnificus cell colonization from LDPE and PP. However, most biofilm biomass, biofilm cell and dispersal cell densities of both species greatly varied after exposure to elevated temperature, pH, and nutrient starvation. It was also found that certain exposures to simulated human media affected both V. parahaemolyticus and V. vulnificus biofilm biomass and biofilm cell densities on LDPE, PP and PS compared to exposure to traditional media of similar pH. Cyclic-di-GMP was higher in biofilm cells compared to dispersal cells, but exposure to more stressful conditions significantly increased signal concentrations in both biofilm and dispersal states. Taken together, this study suggests that human pathogenic strains of V. parahaemolyticus and V. vulnificus can rapidly disperse with high cell densities from different plastic types in vitro. However, the biofilm dispersal process is highly variable, species specific and dependent on plastic type, especially under different human body related environmental exposures.

Quantifying Bacteriorhodopsin Activity as a Function of its Local Environment with a Raman-Based Assay.

Bacteriorhodopsin (bR) is a transmembrane protein that functions as a light-driven proton pump in halophilic archaea. The bR photocycle has been well-characterized; however, these measurements almost exclusively measured purified bR, outside of its native membrane. To investigate what effect the cellular environment has on the bR photocycle, we have developed a Raman-based assay that can monitor the activity of the bR in a variety of conditions, including in its native membrane. The assay uses two continuous-wave lasers, one to initiate photochemistry and one to monitor bR activity. The excitation leads to the steady-state depletion of ground-state bR, which directly relates to the population of photocycle intermediate states. We have used this assay to monitor bR activity both in vitro and in vivo. Our in vitro measurements confirm that our assay is sensitive to bulk environmental changes reported in the literature. Our in vivo measurements show a decrease in bR activity with increasing extracellular pH for bR in its native membrane. The difference in activity with increasing pH indicates that the native membrane environment affects the function of bR. This assay opens the door to future measurements into understanding how the local environment of this transmembrane protein affects function.

Stimulated Raman versus Inverse Raman: Investigating Depletion Mechanisms for Super-Resolution Raman Microscopy.

Super-resolution fluorescence microscopy has been critical in elucidating the nanoscale structure of biological systems. However, fluorescent labels bring difficulties such as perturbative labeling steps and photobleaching. Thus, label-free super-resolution techniques are of great interest, like our group's 2016 stimulated Raman scattering (SRS) technique, stimulated Raman depletion microscopy (SRDM). Inspired by stimulated emission depletion microscopy, SRDM uses a toroidally shaped beam to deplete the signal formed on the edges of the focal spot, resulting in SRS signal being detected from only a subdiffraction limited region. In initial works, the cause of the depletion was not thoroughly characterized. Here, we conclusively demonstrate suppression mechanisms in SRDM, while also contrasting approaches to super-resolution Raman microscopy on the Stokes and anti-Stokes sides of the spectrum. By monitoring the depletion of both the SRS and inverse Raman scattering (IRS) signal at a range of depletion powers, we observed other four-wave coherent Raman pathways that correspond to the introduction of the femtosecond depletion beam. In addition, we showed the depletion of the IRS signal, paving the way for a super-resolution imaging technique based on IRS, inverse raman depletion microscopy (IRDM). Combined, SRDM and IRDM offer label-free super-resolution imaging over a large spectral range to accommodate a variety of different sample constraints.

Label-Free Super-Resolution Imaging Techniques.

Biological and material samples contain nanoscale heterogeneities that are unresolvable with conventional microscopy techniques. Super-resolution fluorescence methods can break the optical diffraction limit to observe these features, but they require samples to be fluorescently labeled. Over the past decade, progress has been made toward developing super-resolution techniques that do not require the use of labels. These label-free techniques span a variety of different approaches, including structured illumination, transient absorption, infrared absorption, and coherent Raman spectroscopies. Many draw inspiration from widely successful fluorescence-based techniques such as stimulated emission depletion (STED) microscopy, photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). In this review, we discuss the progress made in these fields along with the current challenges and prospects in reaching resolutions comparable to those achieved with fluorescence-based methods.

Vibrio parahaemolyticus and Vibrio vulnificus in vitro colonization on plastics influenced by temperature and strain variability.

Marine bacteria often exist in biofilms as communities attached to surfaces, like plastic. Growing concerns exist regarding marine plastics acting as potential vectors of pathogenic Vibrio, especially in a changing climate. It has been generalized that Vibrio vulnificus and Vibrio parahaemolyticus often attach to plastic surfaces. Different strains of these Vibrios exist having different growth and biofilm-forming properties. This study evaluated how temperature and strain variability affect V. parahaemolyticus and V. vulnificus biofilm formation and characteristics on glass (GL), low-density polyethylene (LDPE), polypropylene (PP), and polystyrene (PS). All strains of both species attached to GL and all plastics at 25, 30, and 35°C. As a species, V. vulnificus produced more biofilm on PS (p ≤ 0.05) compared to GL, and biofilm biomass was enhanced at 25°C compared to 30° (p ≤ 0.01) and 35°C (p ≤ 0.01). However, all individual strains' biofilm biomass and cell densities varied greatly at all temperatures tested. Comparisons of biofilm-forming strains for each species revealed a positive correlation (r = 0.58) between their dry biomass weight and OD570 values from crystal violet staining, and total dry biofilm biomass for both species was greater (p ≤ 0.01) on plastics compared to GL. It was also found that extracellular polymeric substance (EPS) chemical characteristics were similar on all plastics of both species, with extracellular proteins mainly contributing to the composition of EPS. All strains were hydrophobic at 25, 30, and 35°C, further illustrating both species' affinity for potential attachment to plastics. Taken together, this study suggests that different strains of V. parahaemolyticus and V. vulnificus can rapidly form biofilms with high cell densities on different plastic types in vitro. However, the biofilm process is highly variable and is species-, strain-specific, and dependent on plastic type, especially under different temperatures.

Beyond single crystals: Imaging rubrene polymorphism across crystalline batches through lattice phonon Raman microscopy.

Polymorphism is an issue troubling numerous scientific fields. A phenomenon where molecules can arrange in different orientations in a crystal lattice, polymorphism in the field of organic photovoltaic materials can dramatically change electronic properties of these materials. Rubrene is a benchmark photovoltaic material showing high carrier mobility in only one of its three polymorphs. To use rubrene in devices, it is important to quantify the polymorph distribution arising from a particular crystal growth method. However, current methods for characterizing polymorphism are either destructive or inefficient for batch scale characterization. Lattice phonon Raman spectroscopy has the ability to distinguish between polymorphs based on low frequency intermolecular vibrations. We present here the addition of microscopy to lattice phonon Raman spectroscopy, which allows us to not only characterize polymorphs efficiently and nondestructively through Raman spectroscopy but also concurrently gain information on the size and morphology of the polymorphs. We provide examples for how this technique can be used to perform large, batch scale polymorph characterization for crystals grown from solution and physical vapor transport. We end with a case study showing how Raman microscopy can be used to efficiently optimize a green crystal growth method, selecting for large orthorhombic crystals desired for rubrene electronic device applications.