Open Access Subvention Fund Articles

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Bioactive Cellulose Nanocrystal-Poly(epsilon-Caprolactone) Nanocomposites for Bone Tissue Engineering Applications
Hong, Jung Ki; Cooke, Shelley L.; Whittington, Abby R.; Roman, Maren (2021-02-25)
3D-printed bone scaffolds hold great promise for the individualized treatment of critical-size bone defects. Among the resorbable polymers available for use as 3D-printable scaffold materials, poly(epsilon-caprolactone) (PCL) has many benefits. However, its relatively low stiffness and lack of bioactivity limit its use in load-bearing bone scaffolds. This study tests the hypothesis that surface-oxidized cellulose nanocrystals (SO-CNCs), decorated with carboxyl groups, can act as multi-functional scaffold additives that (1) improve the mechanical properties of PCL and (2) induce biomineral formation upon PCL resorption. To this end, an in vitro biomineralization study was performed to assess the ability of SO-CNCs to induce the formation of calcium phosphate minerals. In addition, PCL nanocomposites containing different amounts of SO-CNCs (1, 2, 3, 5, and 10 wt%) were prepared using melt compounding extrusion and characterized in terms of Young's modulus, ultimate tensile strength, crystallinity, thermal transitions, and water contact angle. Neither sulfuric acid-hydrolyzed CNCs (SH-CNCs) nor SO-CNCs were toxic to MC3T3 preosteoblasts during a 24 h exposure at concentrations ranging from 0.25 to 3.0 mg/mL. SO-CNCs were more effective at inducing mineral formation than SH-CNCs in simulated body fluid (1x). An SO-CNC content of 10 wt% in the PCL matrix caused a more than 2-fold increase in Young's modulus (stiffness) and a more than 60% increase in ultimate tensile strength. The matrix glass transition and melting temperatures were not affected by the SO-CNCs but the crystallization temperature increased by about 5.5 degrees C upon addition of 10 wt% SO-CNCs, the matrix crystallinity decreased from about 43 to about 40%, and the water contact angle decreased from 87 to 82.6 degrees. The abilities of SO-CNCs to induce calcium phosphate mineral formation and increase the Young's modulus of PCL render them attractive for applications as multi-functional nanoscale additives in PCL-based bone scaffolds.
Computational study of HIV gp120 as a target for polyanionic entry inhibitors: Exploiting the V3 loop region
Hollingsworth, Louis R. IV; Brown, Anne M.; Gandour, Richard D.; Bevan, David R. (PLOS, 2018-01-18)
Multiple approaches are being utilized to develop therapeutics to treat HIV infection. One approach is designed to inhibit entry of HIV into host cells, with a target being the viral envelope glycoprotein, gp120. Polyanionic compounds have been shown to be effective in inhibiting HIV entry, with a mechanism involving electrostatic interactions with the V3 loop of gp120 being proposed. In this study, we applied computational methods to elucidate molecular interactions between the repeat unit of the precisely alternating polyanion, Poly(4,40-stilbenedicarboxylate- alt±maleic acid) (DCSti-alt-MA) and the V3 loop of gp120 from strains of HIV against which these polyanions were previously tested (IIIb, BaL, 92UG037, JR-CSF) as well as two strains for which gp120 crystal structures are available (YU2, 2B4C). Homology modeling was used to create models of the gp120 proteins. Using monomers of the gp120 protein, we applied extensive molecular dynamics simulations to obtain dominant morphologies that represent a variety of open-closed states of the V3 loop to examine the interaction of 112 ligands of the repeating units of DCSti-alt-MA docked to the V3 loop and surrounding residues. Using the distance between the V1/V2 and V3 loops of gp120 as a metric, we revealed through MD simulations that gp120 from the lab-adapted strains (BaL and IIIb), which are more susceptible to inhibition by DCSti-alt-MA, clearly transitioned to the closed state in one replicate of each simulation set, whereas none of the replicates from the Tier II strains (92UG037 and JR-CSF) did so. Docking repeat unit microspecies to the gp120 protein before and after MD simulation enabled identification of residues that were key for binding. Notably, only a few residues were found to be important for docking both before and after MD simulation as a result of the conformational heterogeneity provided by the simulations. Consideration of the residues that were consistently involved in interactions with the ligand revealed the importance of both hydrophilic and hydrophobic moieties of the ligand for effective binding. The results also suggest that polymers of DCSti-alt-MA with repeating units of different configurations may have advantages for therapeutic efficacy.
Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR–Cas9 plasmid
Jo, Ami; Ringel-Scaia, Veronica M.; McDaniel, Dylan K.; Thomas, Cassidy A.; Zhang, Rui; Riffle, Judy S.; Allen, Irving C.; Davis, Richey M. (2020-01-20)
Background The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein system is a revolutionary tool for gene therapy. Despite promising reports of the utility of CRISPR–Cas9 for in vivo gene editing, a principal problem in implementing this new process is delivery of high molecular weight DNA into cells. Results Using poly(lactic-co-glycolic acid) (PLGA), a nanoparticle carrier was designed to deliver a model CRISPR–Cas9 plasmid into primary bone marrow derived macrophages. The engineered PLGA-based carriers were approximately 160 nm and fluorescently labeled by encapsulation of the fluorophore 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS pentacene). An amine-end capped PLGA encapsulated 1.6 wt% DNA, with an encapsulation efficiency of 80%. Release studies revealed that most of the DNA was released within the first 24 h and corresponded to ~ 2–3 plasmid copies released per nanoparticle. In vitro experiments conducted with murine bone marrow derived macrophages demonstrated that after 24 h of treatment with the PLGA-encapsulated CRISPR plasmids, the majority of cells were positive for TIPS pentacene and the protein Cas9 was detectable within the cells. Conclusions In this work, plasmids for the CRISPR–Cas9 system were encapsulated in nanoparticles comprised of PLGA and were shown to induce expression of bacterial Cas9 in murine bone marrow derived macrophages in vitro. These results suggest that this nanoparticle-based plasmid delivery method can be effective for future in vivo applications of the CRISPR–Cas9 system.
Functionalized Cellulose Nanocrystal Nanocomposite Membranes with Controlled Interfacial Transport for Improved Reverse Osmosis Performance
Smith, Ethan D.; Hendren, Keith D.; Haag, James; Foster, Earl Johan; Martin, Stephen M. (MDPI, 2019-01-20)
Thin-film nanocomposite membranes (TFNs) are a recent class of materials that use nanoparticles to provide improvements over traditional thin-film composite (TFC) reverse osmosis membranes by addressing various design challenges, e.g., low flux for brackish water sources, biofouling, etc. In this study, TFNs were produced using as-received cellulose nanocrystals (CNCs) and 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose nanocrystals (TOCNs) as nanoparticle additives. Cellulose nanocrystals are broadly interesting due to their high aspect ratios, low cost, sustainability, and potential for surface modification. Two methods of membrane fabrication were used in order to study the effects of nanoparticle dispersion on membrane flux and salt rejection: a vacuum filtration method and a monomer dispersion method. In both cases, various quantities of CNCs and TOCNs were incorporated into a polyamide TFC membrane via in-situ interfacial polymerization. The flux and rejection performance of the resulting membranes was evaluated, and the membranes were characterized via attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The vacuum filtration method resulted in inconsistent TFN formation with poor nanocrystal dispersion in the polymer. In contrast, the dispersion method resulted in more consistent TFN formation with improvements in both water flux and salt rejection observed. The best improvement was obtained via the monomer dispersion method at 0.5 wt% TOCN loading resulting in a 260% increase in water flux and an increase in salt rejection to 98.98 ± 0.41% compared to 97.53 ± 0.31% for the plain polyamide membrane. The increased flux is attributed to the formation of nanochannels at the interface between the high aspect ratio nanocrystals and the polyamide matrix. These nanochannels serve as rapid transport pathways through the membrane, and can be used to tune selectivity via control of particle/polymer interactions.
Identification of soil bacteria capable of utilizing a corn ethanol fermentation byproduct
Packard, Holly; Taylor, Zachary W.; Williams, Stephanie L.; Guimarães, Pedro Ivo; Toth, Jackson; Jensen, Roderick V.; Senger, Ryan S.; Kuhn, David D.; Stevens, Ann M. (PLoS, 2019-03-08)
A commercial corn ethanol production byproduct (syrup) was used as a bacterial growth medium with the long-term aim to repurpose the resulting microbial biomass as a protein supplement in aquaculture feeds. Anaerobic batch reactors were used to enrich for soil bacteria metabolizing the syrup as the sole nutrient source over an eight-day period with the goal of obtaining pure cultures of facultative organisms from the reactors. Amplification of the V4 variable region of the 16S rRNA gene was performed using barcoded primers to track the succession of microbes enriched for during growth on the syrup. The resulting PCR products were sequenced using Illumina MiSeq protocols, analyzed via the program QIIME, and the alpha-diversity was calculated. Seven bacterial families were the most prevalent in the bioreactor community after eight days of enrichment: Clostridiaceae, Alicyclobacillaceae, Ruminococcaceae, Burkholderiaceae, Bacillaceae, Veillonellaceae, and Enterobacteriaceae. Pure culture isolates obtained from the reactors, and additional laboratory stock strains, capable of facultative growth, were grown aerobically in microtiter plates with the syrup substrate to monitor growth yield. Reactor isolates of interest were identified at a species level using the full 16S rRNA gene and other biomarkers. Bacillus species, commonly used as probiotics in aquaculture, showed the highest biomass yield of the monocultures examined. Binary combinations of monocultures yielded no apparent synergism between organisms, suggesting competition for nutrients instead of cooperative metabolite conversion. © 2019 Packard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Impact of surface topography on biofilm formation by Candida albicans
Lagree, Katherine; Mon, Htwe H.; Mitchell, Aaron P.; Ducker, William A. (PLOS, 2018-06-18)
Candida albicans is a fungal pathogen that causes serious biofilm-based infections. Here we have asked whether surface topography may affect C. albicans biofilm formation. We tested biofilm growth of the prototypical wild-type strain SC5314 on a series of polydimethylsiloxane (PDMS) solids. The surfaces were prepared with monolayer coatings of monodisperse spherical silica particles that were fused together into a film using silica menisci. The surface topography was varied by varying the diameter of the silica particles that were used to form the film. Biofilm formation was observed to be a strong function of particle size. In the particle size range 4.0±8.0 μm, there was much more biofilm than in the size range 0.5± 2.0 μm. The behavior of a clinical isolate from a clade separate from SC5314, strain p76067, showed results similar to that of SC5314. Our results suggest that topographic coatings may be a promising approach to reduce C. albicans biofilm infections.
Mitigation of bidirectional solute flux in forward osmosis via membrane surface coating of zwitterion functionalized carbon nanotubes
Zou, Shiqiang; Smith, Ethan D.; Lin, Shihong; Martin, Stephen M.; He, Zhen (Elsevier, 2019-07-08)
Forward osmosis (FO) has emerged as a promising membrane technology to yield high-quality reusable water from various water sources. A key challenge to be solved is the bidirectional solute flux (BSF), including reverse solute flux (RSF) and forward solute flux (FSF). Herein, zwitterion functionalized carbon nanotubes (Z-CNTs) have been coated onto a commercial thin film composite (TFC) membrane, resulting in BSF mitigation via both electrostatic repulsion forces induced by zwitterionic functional groups and steric interactions with CNTs. At a coating density of 0.97 gm⁻², a significantly reduced specific RSF was observed for multiple draw solutes, including NaCl (55.5% reduction), NH₄H₂PO₄(83.8%), (NH₄)₂HPO₄ (74.5%), NH₄Cl (70.8%), and NH₄HCO₃ (61.9%). When a synthetic wastewater was applied as the feed to investigate membrane rejection, FSF was notably reduced by using the coated membrane with fewer pollutants leaked to the draw solution, including NH₄⁺-N (46.3% reduction), NO₂⁻₋N (37.0%), NO₂⁻₋N (30.3%), K⁺ (56.1%), PO₄³⁻₋P (100%), and Mg²⁺ (100%). When fed with real wastewater, a consistent water flux was achieved during semi-continuous operation with enhanced fouling resistance. This study is among the earliest efforts to address BSF control via membrane modification, and the results will encourage further exploration of effective strategies to reduce BSF.
On-chip manufacturing of synthetic proteins for point-of-care therapeutics
Murphy, Travis W.; Sheng, Jiayuan; Naler, Lynette B.; Feng, Xueyang; Lu, Chang (Nature, 2019)
Therapeutic proteins have recently received increasing attention because of their clinical potential. Currently, most therapeutic proteins are produced on a large scale using various cell culture systems. However, storing and transporting these therapeutic proteins at low temperatures makes their distribution expensive and problematic, especially for applications in remote locations. To this end, an emerging solution is to use point-of-care technologies that enable immediate and accessible protein production at or near the patient’s bedside. Here we present the development of “Therapeutics-On-a-Chip (TOC)”, an integrated microfluidic platform that enables point-of-care synthesis and purification of therapeutic proteins. We used fresh and lyophilized materials for cell-free synthesis of therapeutic proteins on microfluidic chips and applied immunoprecipitation for highly efficient, on-chip protein purification. We first demonstrated this approach by expressing and purifying a reporter protein, green fluorescent protein. Next, we used TOC to produce cecropin B, an antimicrobial peptide that is widely used to control biofilmassociated diseases. We successfully synthesized and purified cecropin B at 63 ng/μl within 6 h with a 92% purity, followed by confirming its antimicrobial functionality using a growth inhibition assay. Our TOC technology provides a new platform for point-of-care production of therapeutic proteins at a clinically relevant quantity.
Raman chemometric urinalysis (Rametrix) as a screen for bladder cancer
Huttanus, Herbert M.; Vu, Tommy; Guruli, Georgi; Tracey, Andrew; Carswell, William; Said, Neveen; Du, Pang; Parkinson, Bing G.; Orlando, Giuseppe; Robertson, John L.; Senger, Ryan S. (2020-08-21)
Bladder cancer (BCA) is relatively common and potentially recurrent/progressive disease. It is also costly to detect, treat, and control. Definitive diagnosis is made by examination of urine sediment, imaging, direct visualization (cystoscopy), and invasive biopsy of suspect bladder lesions. There are currently no widely-used BCA-specific biomarker urine screening tests for early BCA or for following patients during/after therapy. Urine metabolomic screening for biomarkers is costly and generally unavailable for clinical use. In response, we developed Raman spectroscopy-based chemometric urinalysis (Rametrix (TM)) as a direct liquid urine screening method for detecting complex molecular signatures in urine associated with BCA and other genitourinary tract pathologies. In particular, the Rametrix(TM)screen used principal components (PCs) of urine Raman spectra to build discriminant analysis models that indicate the presence/absence of disease. The number of PCs included was varied, and all models were cross-validated by leave-one-out analysis. In Study 1 reported here, we tested the Rametrix (TM) screen using urine specimens from 56 consented patients from a urology clinic. This proof-of-concept study contained 17 urine specimens with active BCA (BCA-positive), 32 urine specimens from patients with other genitourinary tract pathologies, seven specimens from healthy patients, and the urinalysis control Surine(TM). Using a model built with 22 PCs, BCA was detected with 80.4% accuracy, 82.4% sensitivity, 79.5% specificity, 63.6% positive predictive value (PPV), and 91.2% negative predictive value (NPV). Based on the number of PCs included, we found the Rametrix(TM)screen could be fine-tuned for either high sensitivity or specificity. In other studies reported here, Rametrix(TM)was also able to differentiate between urine specimens from patients with BCA and other genitourinary pathologies and those obtained from patients with end-stage kidney disease (ESKD). While larger studies are needed to improve Rametrix(TM)models and demonstrate clinical relevance, this study demonstrates the ability of the Rametrix(TM)screen to differentiate urine of BCA-positive patients. Molecular signature variances in the urine metabolome of BCA patients included changes in: phosphatidylinositol, nucleic acids, protein (particularly collagen), aromatic amino acids, and carotenoids.
Spectral characteristics of urine specimens from healthy human volunteers analyzed using Raman chemometric urinalysis (Rametrix)
Senger, Ryan S.; Kavuru, Varun; Sullivan, Meaghan; Gouldin, Austin; Lundgren, Stephanie; Merrifield, Kristen; Steen, Caitlin; Baker, Emily; Vu, Tommy; Agnor, Ben; Martinez, Gabrielle; Coogan, Hannah; Carswell, William; Karageorge, Lampros; Dev, Devasmita; Du, Pang; Sklar, Allan; Orlando, Giuseppe; Pirkle, James, Jr.; Robertson, John L. (PLOS, 2019-09-27)
Raman chemometric urinalysis (Rametrix™) was used to analyze 235 urine specimens from healthy individuals. The purpose of this study was to establish the “range of normal” for Raman spectra of urine specimens from healthy individuals. Ultimately, spectra falling outside of this range will be correlated with kidney and urinary tract disease. Rametrix™ analysis includes direct comparisons of Raman spectra but also principal component analysis (PCA), discriminant analysis of principal components (DAPC) models, multivariate statistics, and it is available through GitHub as the Rametrix™ LITE Toolbox for MATLAB®. Results showed consistently overlapping Raman spectra of urine specimens with significantly larger variances in Raman shifts, found by PCA, corresponding to urea, creatinine, and glucose concentrations. A 2-way ANOVA test found that age of the urine specimen donor was statistically significant (p < 0.001) and donor sex (female or male identification) was less so (p = 0.0526). With DAPC models and blind leave-one-out build/test routines using the Rametrix™ PRO Toolbox (also available through GitHub), an accuracy of 71% (sensitivity = 72%; specificity = 70%) was obtained when predicting whether a urine specimen from a healthy unknown individual was from a female or male donor. Finally, from female and male donors (n = 4) who contributed first morning void urine specimens each day for 30 days, the co-occurrence of menstruation was found statistically insignificant to Rametrix™ results (p = 0.695). In addition, Rametrix™ PRO was able to link urine specimens with the individual donor with an average of 78% accuracy. Taken together, this study established the range of Raman spectra that could be expected when obtaining urine specimens from healthy individuals and analyzed by Rametrix™ and provides the methodology for linking results with donor characteristics.
Temperature-Dependent Gas Transport Behavior in Cross-Linked Liquid Crystalline Polyacrylate Membranes
Rabie, Feras; Poláková, Lenka; Fallas, Sebastian; Sedlakova, Zdenka; Marand, Eva; Martin, Stephen M. (MDPI, 2019-08-20)
Stable, cross-linked, liquid crystalline polymer (LCP) films for membrane separation applications have been fabricated from the mesogenic monomer 11-(4-cyanobiphenyl-4′-yloxy) undecyl methacrylate (CNBPh), non-mesogenic monomer 2-ethylhexyl acrylate (2-EHA), and cross-linker ethylene glycol dimethacrylate (EGDMA) using an in-situ free radical polymerization technique with UV initiation. The phase behavior of the LCP membranes was characterized using differential scanning calorimetry (DSC) and X-ray scattering, and indicated the formation of a nematic liquid crystalline (LC) phase above the glass transition temperature. The single gas transport behavior of CO₂, CH₄, propane, and propylene in the cross-linked LCP membranes was investigated for a range of temperatures in the LC mesophase and the isotropic phase. Solubility of the gases was dependent not only on the condensability in the LC mesophase, but also on favorable molecular interactions of penetrant gas molecules exhibiting a charge separation, such as CO₂ and propylene, with the ordered polar mesogenic side chains of the LCP. Selectivities for various gas pairs generally decreased with increasing temperature and were discontinuous across the nematic–sotropic transition. Sorption behavior of CO₂ and propylene exhibited a significant change due to a decrease in favorable intermolecular interactions in the disordered isotropic phase. Higher cross-link densities in the membrane generally led to decreased selectivity at low temperatures when the main chain motion was limited by the lack of mesogen mobility in the ordered nematic phase. However, at higher temperatures, increasing the cross-link density increased selectivity as the cross-links acted to limit chain mobility. Mixed gas permeation measurements for propylene and propane showed close agreement with the results of the single gas permeation experiments.

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