The Effect of Bismuth and Tin on Methane and Acetate Production in a Microbial Electrosynthesis Cell Fed with Carbon Dioxide.

This study investigates the impacts of bismuth and tin on the production of CH4 and volatile fatty acids in a microbial electrosynthesis cell with a continuous CO2 supply. First, the impact of several transition metal ions (Ni2+, Fe2+, Cu2+, Sn2+, Mn2+, MoO42-, and Bi3+) on hydrogenotrophic and acetoclastic methanogenic microbial activity was evaluated in a series of batch bottle tests incubated with anaerobic sludge and a pre-defined concentration of dissolved transition metals. While Cu is considered a promising catalyst for the electrocatalytic conversion of CO2 to short chain fatty acids such as acetate, its presence as a Cu2+ ion was demonstrated to significantly inhibit the microbial production of CH4 and acetate. At the same time, CH4 production increased in the presence of Bi3+ (0.1 g L-1) and remained unchanged at the same concentration of Sn2+. Since Sn is of interest due to its catalytic properties in the electrochemical CO2 conversion, Bi and Sn were added to the cathode compartment of a laboratory-scale microbial electrosynthesis cell (MESC) to achieve an initial concentration of 0.1 g L-1. While an initial increase in CH4 (and acetate for Sn2+) production was observed after the first injection of the metal ions, after the second injection, CH4 production declined. Acetate accumulation was indicative of the reduced activity of acetoclastic methanogens, likely due to the high partial pressure of H2. The modification of a carbon-felt electrode by the electrodeposition of Sn metal on its surface prior to cathode inoculation with anaerobic sludge showed a doubling of CH4 production in the MESC and a lower concentration of acetate, while the electrodeposition of Bi resulted in a decreased CH4 production.

Effect of Surface Functionality on the Rheological and Self-Assembly Properties of Chitin and Chitosan Nanocrystals and Use in Biopolymer Films.

Chitin nanocrystals (ChNCs) are unique to all other bio-derived nanomaterials in one aspect: the inherent presence of a nitrogen moiety. By tuning the chemical functionality of this nanomaterial, and thus its charge and hydrogen bonding capacity, one can heavily impact its macroscopic properties such as its rheological and self-assembly characteristics. In this study, two types of ChNCs are made using acid hydrolysis (AH-ChNCs) and oxidative (OX-ChNCs) pathways, unto which deacetylation using a solvent-free procedure is utilized to create chitosan nanocrystals (ChsNCs) of varying degree of deacetylation (DDA). These nanocrystals were then studied for their rheological behavior and liquid crystalline ordering. It was found that with both deacetylation and carboxylation of ChNCs, viscosity continually increased with increasing concentrations from 2 to 8 wt %, contrary to AH-ChNC dispersions in the same range. Interestingly, increasing the amine content of ChNCs was not proportional to the storage modulus, where a peak saturation of amines provided the most stiffness. Conversely, while the introduction of carboxylation increased the elastic modulus of OX-ChNCs by an order of magnitude from that of AH-ChNCs, it was decreased by increasing DDA. Deacetylation and carboxylation both inhibited the formation of a chiral nematic phase. Finally, these series of nanocrystals were incorporated into biodegradable pectin-alginate films as a physical reinforcement, which showed increased tensile strength and Young's modulus values for the films incorporated with ChsNCs. Overall, this study is the first to investigate how surface functionalization of chitin-derived nanocrystals can affect their rheological and liquid crystalline properties and how it augments pectin/alginate films as a physical reinforcement nanofiller.

High-Humidity Shaker Aging to Access Chitin and Cellulose Nanocrystals.

To unlock nature's potential for functional biomaterials, many efforts have been devoted to isolating the nanocrystalline domains within the supramolecular structure of polysaccharides. Yet, low reactivity and yield in aqueous systems along with excessive solvent usage hinders its development. In this report, the first solvent-free pathway to access carboxylated chitin and cellulose nanocrystals with excellent mass balance is described, relying on a new method coined high-humidity shaker aging (HHSA). The method involves a mild grinding of the polysaccharide with ammonium persulfate followed by an aging phase under high-humidity and on a shaker plate. Insights into the mechanism were uncovered, which highlighted the unique role of high humidity to afford a gradual uptake of water by the material up to deliquescence when the reaction is complete. This process was then validated for direct synthesis of nanocrystals from biomass sources including crab and soft wood pulp.

Carboxylated Chitosan Nanocrystals: A Synthetic Route and Application as Superior Support for Gold-Catalyzed Reactions.

In this study, we demonstrate for the first time the fabrication of carboxylated chitosan nanocrystals (ChsNC) with high degree of deacetylation (DDA) at >80% and narrow size distribution. We also studied its application as a sustainable support material for metal-based catalysts. Carboxylated chitin nanocrystals (ChNCs) were initially prepared through partial cleavage of glycosidic bonds in chitin by ammonium persulfate, with concurrent oxidation of chitin C6 primary alcohols to produce carboxylate groups on the surface of the ChNCs. ChsNCs were subsequently prepared using an alkaline deacetylation procedure in the presence of NaBH4 to preserve the nanorod structure of the biomaterial. The resulting nanocrystals feature both carboxyl and amino functional groups. Transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier-transform infrared (FTIR) spectroscopy were used to determine the morphology and composition of these carboxylated ChNCs and ChsNCs. Subsequently, we tested the ability of the as-made ChsNCs as a biomass-based catalyst support for Au nanoparticles (NPs) using the 4-nitrophenol reduction and the aldehyde-amine-alkyne (A3) coupling reactions to demonstrate its capabilities in regard to the ones of cellulose nanocrystals (CNCs). In particular, Au NPs over ChsNCs featured the highest turnover frequency (TOF) value for the 4-nitrophenol reduction reported for all Au-based catalysts supported on carbon-based systems. Spectroscopic and imaging techniques confirmed the importance of precisely controlling the redox state of Au as it is being deposited to afford a highly disperse active site on the bionano-support.

Palladium nanoparticles supported on chitin-based nanomaterials as heterogeneous catalysts for the Heck coupling reaction.

In this report, chitin and chitosan nanocrystals were used as biomass-based supports for Pd nanoparticles (NPs) used as a heterogeneous catalyst for the Heck coupling reaction. By using a one-pot fabrication method, a Pd salt precursor was directly reduced and deposited onto these nanocrystal catalysts. Characterization of these nanocomposites showed disperse Pd NPs on the surfaces of the chitinous nanocrystals. Heck coupling model reactions revealed full product yield in relatively benign conditions, outcompeting the use of other catalysts supported on biomass-based nanomaterials, including cellulose nanocrystals. These initial results show the potential for using chitinous nanomaterials as effective catalyst supports in cross-coupling reactions.

Fabrication of thermo-responsive multicore microcapsules using a facile extrusion process.

A process employing extrusion was used to produce multicore microcapsules composed of multiple beads. The inner beads were made from κ-carrageenan (κ-c), a thermo-responsive linear sulphated polymer whose gelling temperature ranges at 40-60 °C, depending on the concentration of κ-c polymer and the amount of potassium chloride used for gelation. The resulting beads were then enveloped by chitosan through gelation with sodium triphosphate. The pesticide ammonium glufosinate was encapsulated in the κ-c/chitosan multicore microcapsules for demonstration of controlled release of the encapsulant. It was found that in response to an external stimulus, such as elevated temperature or solar simulation, the microcapsules exhibit the gradual release of encapsulated pesticide molecules from multicore microcapsules, compared with beads only. This process of making multicore microcapsules can be extended to other polymer pairs based on applications. This work is relevant to agriculture, where the controlled-release of the pesticides or fertilizers could be triggered by the sun and/or temperature changes, thus extending the residual period of the chemicals as well as decreasing the extent of pollution by leaching of abundant chemicals.

Production, purification and immunogenicity of Gag virus-like particles carrying SARS-CoV-2 components.

The virus-like particle (VLP) platform is a robust inducer of humoral and cellular immune responses; hence, it has been used in vaccine development for several infectious diseases. In the current work, VLPs carrying SARS-CoV-2 Spike (S) protein (Wuhan strain) with an HIV-1 Gag core were produced using suspension HEK 293SF-3F6 cells by transient transfection. The Gag was fused with green fluorescent protein (GFP) for rapid quantification of the VLPs. Five different versions of Gag-Spike VLPs (Gag-S-VLPs) consisting of Gag-S alone or combined with other SARS-CoV-2 components, namely Gag-S-Nucleocapsid (N), Gag-S-Matrix (M), Gag-S-Envelope (E), Gag-S-MEN, along with Gag alone were produced and processed by clarification, nuclease treatment, concentration by tangential flow filtration (TFF) and diafiltration. A pilot mouse study was performed to evaluate the immunogenicity of the Gag-S-VLPs through the measurement of the humoral and/or cellular responses against all the mentioned SARS-CoV-2 components. Antibody response to Spike was observed in all variants. The highest number of Spike-specific IFN-γ + T cells was detected with Gag-S-VLPs. No induction of antigen-specific cellular responses to M, N or E proteins were detected with any of the Gag-S, M, E/or N VLPs tested. Therefore, the Gag-S-VLP, by reason of consistently eliciting strong antigen-specific cellular and antibody responses, was selected for further evaluation. The purification process was improved by replacing the conventional centrifugation by serial microfiltration in the clarification step, followed by Spike-affinity chromatography to get concentrated VLPs with higher purity. Three different doses of Gag-S-VLP in conjunction with two adjuvants (Quil-A or AddaVax) were used to assess the dose-dependent antigen-specific cellular and antibody responses in mice. The Gag-S-VLP adjuvanted with Quil-A resulted in a stronger Spike-specific cellular response compared to that adjuvanted with AddaVax. A strong spike neutralisation activity was observed for all doses, independent of the adjuvant combination.

Exploring the functional attributes and in vitro starch and protein digestibility of pea flours having a wide range of amylose content.

In this study, ten pea flours covering a broad range of amylose content (37.2-77.6 %, dsb) were characterized for functional and nutritional properties. As the amylose contents increased, the starch contents of the pea flours showed a downward trend (r = -0.990, p < 0.001 in Pearson correlation) but their protein and total dietary fiber contents exhibited an upward trend (r = 0.915, p < 0.001 and r = 0.885, p < 0.001, respectively). A greater amylose content tended to increase starch gelatinization temperatures of the pea flours, which thus required a higher cooking temperature for pasting viscosity development and subsequent gel formation. An increased amylose level reduced in vitro starch digestibility of the cooked pea flours (r = -0.944, p < 0.001) but did not influence in vitro protein digestibility. The insightful findings will be valuable for utilizing the diverse pea lines to create new flour, starch, and protein ingredients.

Carboxylated Cellulose Nanocrystals Decorated with Varying Molecular Weights of Poly(diallyldimethylammonium chloride) as Sustainable Antibacterial Agents.

Cationic nanomaterials are promising candidates for the development of effective antibacterial agents by taking advantage of the nanoscale effects as well as other exceptional physicochemical properties of nanomaterials. In this study, carboxylated cellulose nanocrystals (cCNCs) derived from softwood pulp were coated with cationic poly(diallyldimethylammonium chloride) of varying molecular weights. The resulting cationic carboxylated cellulose nanocrystals coated with poly(diallyldimethylammonium chloride) (cCNCs-PDDA) nanomaterials were characterized for their structural and morphological properties using Fourier transform infrared spectroscopy, dynamic light scattering, zeta potential, elemental analysis, transmission electron microscopy, and thermogravimetric analysis. Cationic cCNCs-PDDA were investigated for their antibacterial properties against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli 23934 and Pseudomonas aeruginosa using a bacterial lawn growth inhibition assay. cCNC-PDDA materials displayed marked antibacterial activity, particularly against Gram-positive Staphylococcus aureus. Overall, our results indicated that cCNCs-PDDA could be a potential candidate for antibacterial applications such as antibacterial surfaces or coatings.

Inducible HEK293 AAV packaging cell lines expressing Rep proteins.

Packaging or producer cell lines for scalable recombinant adeno-associated virus (rAAV) production have been notoriously difficult to create due in part to the cytostatic nature of the Rep proteins required for AAV production. The most difficult challenge being creating AAV packaging cell lines using HEK293 parental cells, currently the best mammalian platform for rAAV production due to the constitutive expression of E1A in HEK293 cells, a key REP transcription activator. Using suspension and serum-free media adapted HEK293SF carrying a gene expression regulation system induced by addition of cumate and coumermycin, we were able to create REP-expressing AAV packaging cells. This was achieved by carefully choosing two of the AAV Rep proteins (Rep 40 and 68), using two inducible promoters with different expression levels and integrating into the cells through lentiviral vector transduction. Three of our best clones produced rAAV titers comparable to titers obtained by standard triple plasmid transfection of their parental cells. These clones were stable for up to 7 weeks under continuous cultures condition. rAAV production from one clone was also validated at scale of 1 L in a wave bioreactor using serum-free suspension culture.

Characterization of Virulent T4-Like Acinetobacter baumannii Bacteriophages DLP1 and DLP2.

The world is currently facing a global health crisis due to the rapid increase in antimicrobial-resistant bacterial infections. One of the most concerning pathogens is Acinetobacter baumannii, which is listed as a Priority 1 pathogen by the World Health Organization. This Gram-negative bacterium has many intrinsic antibiotic resistance mechanisms and the ability to quickly acquire new resistance determinants from its environment. A limited number of effective antibiotics against this pathogen complicates the treatment of A. baumannii infections. A potential treatment option that is rapidly gaining interest is "phage therapy", or the clinical application of bacteriophages to selectively kill bacteria. The myoviruses DLP1 and DLP2 (vB_AbaM-DLP_1 and vB_AbaM-DLP_2, respectively) were isolated from sewage samples using a capsule minus variant of A. baumannii strain AB5075. Host range analysis of these phages against 107 A. baumannii strains shows a limited host range, infecting 15 and 21 for phages DLP1 and DLP2, respectively. Phage DLP1 has a large burst size of 239 PFU/cell, a latency period of 20 min, and virulence index of 0.93. In contrast, DLP2 has a smaller burst size of 24 PFU/cell, a latency period of 20 min, and virulence index of 0.86. Both phages show potential for use as therapeutics to combat A. baumannii infections.

Preclinical evaluation of manufacturable SARS-CoV-2 spike virus-like particles produced in Chinese Hamster Ovary cells.

BACKGROUND: As the COVID-19 pandemic continues to evolve, novel vaccines need to be developed that are readily manufacturable and provide clinical efficacy against emerging SARS-CoV-2 variants. Virus-like particles (VLPs) presenting the spike antigen at their surface offer remarkable benefits over other vaccine antigen formats; however, current SARS-CoV-2 VLP vaccines candidates in clinical development suffer from challenges including low volumetric productivity, poor spike antigen density, expression platform-driven divergent protein glycosylation and complex upstream/downstream processing requirements. Despite their extensive use for therapeutic protein manufacturing and proven ability to produce enveloped VLPs, Chinese Hamster Ovary (CHO) cells are rarely used for the commercial production of VLP-based vaccines. METHODS: Using CHO cells, we aimed to produce VLPs displaying the full-length SARS-CoV-2 spike. Affinity chromatography was used to capture VLPs released in the culture medium from engineered CHO cells expressing spike. The structure, protein content, and glycosylation of spikes in VLPs were characterized by several biochemical and biophysical methods. In vivo, the generation of neutralizing antibodies and protection against SARS-CoV-2 infection was tested in mouse and hamster models. RESULTS: We demonstrate that spike overexpression in CHO cells is sufficient by itself to generate high VLP titers. These VLPs are evocative of the native virus but with at least three-fold higher spike density. In vivo, purified VLPs elicit strong humoral and cellular immunity at nanogram dose levels which grant protection against SARS-CoV-2 infection. CONCLUSIONS: Our results show that CHO cells are amenable to efficient manufacturing of high titers of a potently immunogenic spike protein-based VLP vaccine antigen.

Virus-like particles (VLPs) have a structure that is similar to viruses but they cannot cause infection or illness. If VLPs are injected into the body they produce an immune response similar to that seen following infection by a virus. This means that VLPs can be used as vaccines against viruses that cause illness in people. Many drugs, named biologics, are manufactured using living cells, including cells that were originally derived from Chinese Hamster Ovaries (CHO cells). We developed a simple method to produce VLPs similar to the SARS-CoV-2 virus in CHO cells. We show that vaccination of rodents with these VLPs prevents them from becoming ill following infection with SARS-CoV-2. These VLPs could become a part of an alternative, easily produced vaccine for the prevention of COVID-19 in humans.

Evaluation of biocathode materials for microbial electrosynthesis of methane and acetate.

This study compares carbon felt (CF), granular activated carbon (GAC), and a conductive acrylonitrile butadiene styrene (cABS) polymer cathodes for CH4 and acetate production in a microbial electrosynthesis (MES) cell. At an applied voltage of 2.8 V and continuous CO2 flow, the CF biocathode MES cell showed the highest CH4 production rate of 1420 ± 225 mL Vc-1 d-1 (Vc = cathode volume), also producing acetate at a rate of 710 ± 110 mg Vc-1 d-1. The volumetric rates of acetate and CH4 production decreased when using the GAC cathode (720 ± 94 mL Vc-1 d-1 and 236 ± 65 mg Vc-1 d-1, respectively). When the cABS cathode was used, the CH4 production declined to 250 ± 35 mL Vc-1 d-1, while the acetate production increased to 1105 ± 130 mg Vc-1 d-1. The biocatalytic activity of cABS increased after in-situ electrodeposition of Ni and Fe, resulting in a current increase from 205 mA to 380 mA accompanied by increasing acetate and ethanol production (1405 mg Vc-1 d-1 and 240 mg Vc-1 d-1, respectively), while the CH4 production decreased. The cABS cathode showed the highest specific (per surface area) activity for acetate and CH4 production.

Design of chitosan nanocrystals decorated with amino acids and peptides.

Antimicrobial peptides (AMPs) offer a great promise in designing new therapeutics due to their ability to interfere in bacterial growth by penetrating the cell wall. The overuse of antibiotics has resulted into antibiotic-resistant bacteria and AMPs could be an alternative to circumvent this resistance. Chitosan nanocrystals (ChsNCs) are rod-shaped polysaccharide-based nanomaterials, formed by deacetylation of seafood waste. They possess primary amino groups on the surface of the nanoparticles which can be as used a scaffold due to the built-in morphology and ease in functionalization. Here, we developed a new methodology to functionalize ChsNCs with amino acids and peptides by using fundamentals of solid phase peptide synthesis. The resulting functionalized rod-shaped nanomaterials were characterized using nuclear magnetic resonance (NMR), dynamic light scattering (DLS), zeta potential measurements and microscopy imaging. This synthetic strategy could be used in designing ChsNC-based nanomaterials to target specific cells by attaching bioactive peptides to the nanomaterial surface.

Selenite and selenate removal in a permeable flow-through bioelectrochemical barrier.

This study demonstrated the removal of selenite and selenate in flow-through permeable bioelectrochemical barriers (microbial electrolysis cells, MECs). The bioelectrochemical barriers consisted of cathode and anode electrode compartments filled with granular carbon or metallurgical coke. A voltage of 1.4 V was applied to the electrodes to enable the bioelectrochemical removal of selenium species. For comparison, a similarly designed permeable anaerobic biobarrier filled with granular carbon was operated without voltage. All biobarrier setups were fed with water containing up to 5,000 µg L-1 of either selenite or selenate and 70 mg L-1 of acetate as a source of organic carbon. Significant removal of selenite and selenate was observed in MEC experimental setups, reaching 99.5-99.8% over the course of the experiment, while in the anaerobic biobarrier the removal efficiency did not exceed 88%. By simultaneously operating several setups and changing operating parameters (selenium species, influent Se and acetate concentrations, etc.) we demonstrated enhanced removal of Se species under bioelectrochemical conditions.

Synthesis and Cytotoxicity Studies of Wood-Based Cationic Cellulose Nanocrystals as Potential Immunomodulators.

Polysaccharides have been shown to have immunomodulatory properties. Modulation of the immune system plays a crucial role in physiological processes as well as in the treatment and/or prevention of autoimmune and infectious diseases. Cellulose nanocrystals (CNCs) are derived from cellulose, the most abundant polysaccharide on the earth. CNCs are an emerging class of crystalline nanomaterials with exceptional physico-chemical properties for high-end applications and commercialization prospects. The aim of this study was to design, synthesize, and evaluate the cytotoxicity of a series of biocompatible, wood-based, cationic CNCs as potential immunomodulators. The anionic CNCs were rendered cationic by grafting with cationic polymers having pendant +NMe3 and +NH3 moieties. The success of the synthesis of the cationic CNCs was evidenced by Fourier transform infrared spectroscopy, dynamic light scattering, zeta potential, and elemental analysis. No modification in the nanocrystals rod-like shape was observed in transmission electron microscopy and atomic force microscopy analyses. Cytotoxicity studies using three different cell-based assays (MTT, Neutral Red, and LIVE/DEAD®) and three relevant mouse and human immune cells indicated very low cytotoxicity of the cationic CNCs in all tested experimental conditions. Overall, our results showed that cationic CNCs are suitable to be further investigated as immunomodulators and potential vaccine nanoadjuvants.

Selective nanomolar detection of dopamine using a boron-doped diamond electrode modified with an electropolymerized sulfobutylether-beta-cyclodextrin-doped poly(N-acetyltyramine) and polypyrrole composite film.

N-acetyltyramine was synthesized and electropolymerized together with a negatively charged sulfobutylether-beta-cyclodextrin on a boron-doped diamond (BDD) electrode followed by the electropolymerization of pyrrole to form a stable and permselective film for selective dopamine detection. The selectivity and sensitivity of the formed layer-by-layer film was governed by the sequence of deposition and the applied potential. Raman results showed a decrease in the peak intensity at 1329 cm(-1) (sp(3)), the main feature of BDD, upon each electrodeposition step. Such a decrease was correlated well with the change of the charge-transfer resistance derived from impedance data, i.e., reflecting the formation of the layer-by-layer film. The polycrystalline BDD surface became more even with lower surface roughness as revealed by scanning electron and atomic force microscopy. The modified BDD electrode exhibited rapid response to dopamine within 1.5-2 s and a low detection limit of 4-5 nM with excellent reproducibility. Electroactive interferences caused by 4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, ascorbic acid, and uric acid were completely eliminated, whereas the signal response of epinephrine and norepinephrine was significantly suppressed by the permselective film.

Selective detection of dopamine using a combined permselective film of electropolymerized (poly-tyramine and poly-pyrrole-1-propionic acid) on a boron-doped diamond electrode.

An effective and robust electrochemical approach has been developed for selective detection of dopamine in the presence of 3,4-dihydroxyphenylalanine (l-DOPA), ascorbic acid, uric acid and other dopamine metabolites. A 'layer-by-layer' film of tyramine and pyrrole-1-propionic acid (PPA) was formed by subsequent electropolymerization on a boron-doped diamond (BDD) electrode with an overall thickness of approximately 33 nm as estimated by AFM. The formation of the electropolymerized homogeneous film was also confirmed by SEM and Raman spectroscopy. The modified BDD electrode exhibited rapid response to dopamine within 6 s and a detection limit of 50 nM with excellent reproducibility. The stable electropolymerized film was capable of excluding electroactive interference from 20 microM l-DOPA, 20 microM 3,4-dihydroxyphenylacetic acid (DOPAC), and ascorbic and uric acids at normal physiological conditions (100 microM each). The modified electrode could be used for several repeated analyses of dopamine at 5 microM, without noticeable surface fouling. A plausible mechanism for permselectivity was suggested and supported by pertinent experimental data.

Assessment of cytotoxicity of quantum dots and gold nanoparticles using cell-based impedance spectroscopy.

A continuous online technique based on electric cell-substrate impedance sensing (ECIS) was demonstrated for measuring the concentration and time response function of fibroblastic V79 cells exposed to nanomaterials such as quantum dots (QDs) and fluorescent gold nanoparticles. The half-inhibition concentration, (ECIS50), the required concentration to attain 50% inhibition of the cytotoxic response, was estimated from the response function to ascertain cytotoxicity during the course of measurement. The ECIS50 values agreed well with the results obtained using the standard neutral red assay. Cadmium selenide quantum dots showed direct cytotoxicity with the ECIS assay. For the cadmium telluride quantum dots, significant toxicity could be assigned to free cadmium, although additional toxicity could be attributed to the QDs per se. The QDs synthesized with indium gallium phosphide and the fluorescent gold nanoparticles were not cytotoxic.