RESUMEN

In the glycerolysis process for diacylglycerol (DAG) preparation, free lipases suffer from poor stability and the inability to be reused. To address this, a cost-effective immobilized lipase preparation was developed by cross-linking macroporous resin with poly (ethylene glycol) diglycidyl ether (PEGDGE) followed by lipase adsorption. The selected immobilization conditions were identified as pH 7.0, 35 °C, cross-linking agent concentration 2.0%, cross-linking time 4 h, lipase amount 5 mg/g of support, and adsorption time 4 h. Enzymatic properties of the immobilized lipase were analyzed, revealing enhanced pH stability, thermal stability, storage stability, and operational stability post-immobilization. The conditions for immobilized enzyme-catalyzed glycerolysis to produce DAG were selected, demonstrating the broad applicability of the immobilized lipase. The immobilized lipase catalyzed glycerolysis reactions using various oils as substrates, with DAG content in the products ranging between 35 and 45%, demonstrating broad applicability. Additionally, the changes during the repeated use of the immobilized lipase were characterized, showing that mechanical damage, lipase leakage, and alterations in the secondary structure of the lipase protein contributed to the decline in catalytic activity over time. These findings provide valuable insights for the industrial application of lipase.

Asunto(s)

Diglicéridos , Estabilidad de Enzimas , Enzimas Inmovilizadas , Lipasa , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Lipasa/química , Lipasa/metabolismo , Diglicéridos/química , Concentración de Iones de Hidrógeno , Glicerol/química , Temperatura , Eurotiales/enzimología , Biocatálisis , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo

RESUMEN

Capillary flow profile of liquid samples in porous media is closely related to the important properties of liquid samples, including the viscosity and the surface energy. Therefore, capillary flow profile can be used as an index to differentiate liquid samples with different properties. Fast and automatic characterization of capillary flow profile of liquid samples is necessary. In this work, we develop a portable and economical capacitance acquisition system (CASY) to easily obtain the capillary flow profile of liquid samples on microfluidic thread-based analytical devices (µTADs) by measuring the capacitance during the capillary flow. At first, we validate the accuracy of this method by comparing with the traditional method by video analysis in obtaining the capillary flow profiles in µTADs of cotton threads or glass fiber threads. Then we use it to differentiate liquid samples with different viscosity (mixture of water and glycerol). In addition, capillary flow profile on µTADs with chemical valves (chitosan or sucrose) can also be obtained on this device. Lastly, we show the potential of this device in measurement of hematocrit (HCT) of whole blood samples. This device can be used to catalog liquid biological samples with different properties in point-of-care diagnostics in the near future.

Asunto(s)

Capacidad Eléctrica , Viscosidad , Hematócrito , Técnicas Analíticas Microfluídicas/instrumentación , Diseño de Equipo , Humanos , Dispositivos Laboratorio en un Chip , Agua/química , Glicerol/química

RESUMEN

Eco-friendly materials like carbohydrate-based polymers are important for a sustainable future. Starch is particularly promising because of its biodegradability and abundance but its processing to thermoplastic starch requires optimization. Here we developed thermoplastic maize starch materials based on three manufacturing protocols, namely: (1) starch/glycerol manual mixing and extrusion, (2) starch/glycerol manual mixing, extrusion, and kneading, (3) starch/glycerol/water manual mixing and kneading. The physical properties were investigated by differential scanning calorimetry, thermogravimetric analysis, and broadband dielectric spectroscopy. As expected from a partially miscible blend, the dielectric spectra revealed two distinct α-relaxations for the glycerol-rich and the starch-rich phases, respectively. By employing kneading after extrusion, the miscibility between the two phases was found to improve based on thermal and dielectric methods. Moreover, the addition of water during the premixing stage was observed to facilitate phase separation between starch and glycerol, with the α-relaxation dynamics of the latter being comparable to pure glycerol.

Asunto(s)

Glicerol , Almidón , Zea mays , Almidón/química , Glicerol/química , Zea mays/química , Agua/química , Rastreo Diferencial de Calorimetría , Termogravimetría , Temperatura

RESUMEN

Regenerative medicine is continuously looking for new natural, biocompatible and possibly biodegradable materials, but also mechanically compliant. Chitosan is emerging as a promising FDA-approved biopolymer for tissue engineering, however, its exploitation in regenerative devices is limited by its brittleness and can be further improved, for example by blending it with other materials or by tuning its superficial microstructure. Here, we developed membranes made of chitosan (Chi) and glycerol, by solvent casting, and micro-patterned them with directional geometries having different levels of axial symmetry. These membranes were characterized by light microscopies, atomic force microscopy (AFM), by thermal, mechanical and degradation assays, and also testedin vitroas scaffolds with Schwann cells (SCs). The glycerol-blended Chi membranes are optimized in terms of mechanical properties, and present a physiological-grade Young's modulus (≈0.7 MPa). The directional topographies are effective in directing cell polarization and migration and in particular are highly performant substrates for collective cell migration. Here, we demonstrate that a combination of a soft compliant biomaterial and a topographical micropatterning can improve the integration of these scaffolds with SCs, a fundamental step in the peripheral nerve regeneration process.

Asunto(s)

Materiales Biocompatibles , Movimiento Celular , Quitosano , Módulo de Elasticidad , Glicerol , Regeneración Nerviosa , Células de Schwann , Ingeniería de Tejidos , Andamios del Tejido , Cicatrización de Heridas , Quitosano/química , Células de Schwann/citología , Glicerol/química , Animales , Materiales Biocompatibles/química , Andamios del Tejido/química , Ratas , Ingeniería de Tejidos/métodos , Microscopía de Fuerza Atómica , Ensayo de Materiales , Membranas Artificiales , Medicina Regenerativa/métodos

RESUMEN

In this study, the biosynthesis of polyhydroxyalkanoates (PHAs) was carried out using Pseudomonas putida and Pseudomonas aeruginosa. These PHAs were produced using reagent-grade glycerol and crude glycerol as the carbon sources. The objective was to compare the production of PHAs and to functionalize these polymers with silver nanoparticles to provide antibacterial properties for potential biomedical applications. The findings from the physical and chemical analyses confirmed the successful synthesis and extraction of PHAs, achieving comparable yields using both crude glycerol and reagent-grade glycerol as carbon sources across both strains. Approximately 16% higher PHAs production was obtained using Pseudomonas putida compared to Pseudomonas aeruginosa, and no significant difference was observed in the production rate of PHAs between the two carbon sources used, which means that crude glycerol could be utilized even though it has more impurities. Notably, PHAs functionalized with silver nanoparticles showed improved antibacterial effectiveness, especially those derived from reagent-grade glycerol and the Pseudomonas aeruginosa strain.

Asunto(s)

Antibacterianos , Glicerol , Nanopartículas del Metal , Polihidroxialcanoatos , Pseudomonas aeruginosa , Pseudomonas putida , Plata , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Pseudomonas putida/metabolismo , Plata/química , Plata/farmacología , Polihidroxialcanoatos/biosíntesis , Polihidroxialcanoatos/química , Nanopartículas del Metal/química , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/biosíntesis , Glicerol/química , Glicerol/metabolismo , Pruebas de Sensibilidad Microbiana

RESUMEN

Electronic skin (e-skin) is considered as a highly promising interface for human-computer interaction systems and wearable electronic devices. Through elaborate design and assembly of various materials, it possesses multiple characteristics similar to human skin, including remarkable flexibility, stretchability, sensitivity to temperature and humidity, biocompatibility, and efficient interfacial ion/electron transport capabilities. Here, we innovatively integrate multifunctional carbon quantum dots (CQDs), which exhibit conductivity, antibacterial properties, ultraviolet absorption, and fluorescence emission, with poly(acrylic acid) and glycerin (Gly) into a three-dimensional network structure of natural goatskin collagen fibers. Through a top-down design strategy enhanced by hydrogen bond reconstruction, we successfully fabricated a novel transparent e-skin (PAC-eSkin). This e-skin exhibited significant tensile properties (4.94 MPa of tensile strength and 263.42% of a maximum breaking elongation), while also possessing Young's modulus similar to human skin (2.32 MPa). It is noteworthy that the functionalized CQDs used was derived from discarded goat hair, and the addition of Gly gave PAC-eSkin excellent antifreezing and moisturizing properties. Due to the presence of ultrasmall CQDs, which creates efficient ion/electron transport channels within PAC-eSkin, it could rapidly sense human motion and physiological signals (with a gauge factor (GF) of 1.88). Furthermore, PAC-eSkin had the potential to replace traditional electrode patches for real-time monitoring of electrocardiogram, electromyogram, and electrooculogram signals, with a higher SNR (signal-to-noise ratio) of 25.1 dB. Additionally, the customizable size and shape of PAC-eSkin offer vast possibilities for the construction of single-electrode triboelectric nanogenerator systems. We have reason to believe that the design and development of this transparent e-skin based on CQDs-functionalized dermal collagen matrices can pave a new way for innovations in human-computer interaction interfaces and their sensing application in diverse scenarios.

Asunto(s)

Carbono , Puntos Cuánticos , Dispositivos Electrónicos Vestibles , Puntos Cuánticos/química , Humanos , Carbono/química , Animales , Resinas Acrílicas/química , Glicerol/química , Cabras , Dermis , Resistencia a la Tracción , Colágeno/química , Conductividad Eléctrica

RESUMEN

Dynamic Nuclear Polarization (DNP) is a technique that leverages the quantum sensing capability of electron spins to enhance the sensitivity of nuclear magnetic resonance (NMR) signals, especially for insensitive samples. Glassing agents play a crucial role in the DNP process by facilitating the transfer of polarization from the unpaired electron spins to the nuclear spins along with cryoprotection of biomolecules. DNPjuice comprising of glycerol-d8/D2O/H2O has been extensively used for this purpose over the past two decades. Polyethylene glycol (PEG), also used as a cryoprotectant, is often used as a crowding agent in experimental setups to mimic cellular conditions, particularly the invitro preparation of liquid-liquid phase separated (LLPS) condensates. In this study, we investigate the efficacy of PEG as an alternative to glycerol in the DNP juice, critical for signal enhancement. The modified DNP matrix leads to high DNP enhancement which enables direct study of LLPS condensates by solid-state DNP methods without adding any external constituents. An indirect advantage of employing PEG is that the PEG signals appear at ∼72.5 ppm and are relatively well-separated from the aliphatic region of the protein spectra. Large cross-effect DNP enhancement is attained for 13C-glycine by employing the PEG-water mixture as a glassing agent and ASYMPOL-POK as the state-of-art polarizing agent, without any deuteration. The DNP enhancement and the buildup rates are similar to results obtained with DNP juice, conforming to that PEG serves as a good candidate for both inducing crowding and glassing agent in the study of LLPS.

Asunto(s)

Polietilenglicoles , alfa-Sinucleína , Polietilenglicoles/química , alfa-Sinucleína/química , Resonancia Magnética Nuclear Biomolecular , Glicerol/química , Humanos

RESUMEN

An innovative integrated paper-based microdevice was developed for protein separation by isoelectric focusing (IEF), allowing for robust design thanks to a 3D-printed holder integrating separation channel, reservoirs, and electrodes. To reach robustness and precision, the optimization focused on the holder geometry, the paper nature, the reservoir design, the IEF medium, and various focusing parameters. A well-established and stable pH gradient was obtained on a glass-fiber paper substrate with simple sponge reservoirs, and the integration of the electrodes in the holder led to a straightforward system. The separation medium composed of water/glycerol (85/15, v/v) allowed for reducing medium evaporation while being an efficient medium for most hydrophobic and hydrophilic proteins, compatible with mass spectrometry detection for further proteomics developments. To our knowledge, this is the first report of the use of glycerol solutions as a separation medium in a paper-based microdevice. Analytical performances regarding pH gradient generation, pI determination, separation efficiency, and resolution were estimated while varying the IEF experimental parameters. The overall process led to an efficient separation within 25 min. Then, this methodology was applied to a sample composed of saliva doped with proteins. A minimal matrix effect was evidenced, underscoring the practical viability of our platform. This low-cost, versatile and robust paper-based IEF microdevice opens the way to various applications, ranging from sample pre-treatment to integration in an overall proteomic-on-a-chip device.

Asunto(s)

Glicerol , Focalización Isoeléctrica , Papel , Proteínas , Focalización Isoeléctrica/instrumentación , Focalización Isoeléctrica/métodos , Proteínas/análisis , Proteínas/aislamiento & purificación , Glicerol/química , Glicerol/análisis , Concentración de Iones de Hidrógeno , Diseño de Equipo , Humanos , Dispositivos Laboratorio en un Chip , Saliva/química , Técnicas Analíticas Microfluídicas/instrumentación , Proteómica/métodos , Interacciones Hidrofóbicas e Hidrofílicas

RESUMEN

Viscoelastic relaxation mechanisms of individualized cellulose nanofibers (iCNFs) dispersed in glycerol in the dilute and semidilute regions were investigated by linear viscoelastic and dynamic birefringence measurements. The birefringence relaxation of the iCNFs was described by the orientational and curvature modes of an existing viscoelastic theory for ideal semiflexible polymers (Shankar-Pasquali-Morse theory). However, the Shankar-Pasquali-Morse theory could not fully describe the iCNF viscoelastic relaxation at high frequencies. Considering the results for birefringence relaxation, the experimental tension mode of the iCNFs was evaluated to be higher than the theoretical value. These results show that the viscoelastic relaxations of the iCNFs are different from those of ideal semiflexible polymers, in contrast to cellulose nanocrystals (CNCs). As the iCNF concentration increased, the orientational mode dramatically slowed, which was more drastic than other semiflexible polymers, including CNCs. This anomalous behavior is likely due to the nonideal nature of iCNFs.

Asunto(s)

Celulosa , Elasticidad , Nanofibras , Nanofibras/química , Celulosa/química , Birrefringencia , Viscosidad , Glicerol/química

RESUMEN

Developing utilization technologies for biomass resources, exploring their applications in the fields of energy and chemical engineering, holds significant importance for promoting sustainable development and constructing a green, low-carbon society. In this study, we designed a non-natural in vitro multi-enzyme system for converting glycerol and CO2 into L-aspartic acid (L-Asp). The coupled system utilized eight enzymes, including alditol oxidase (ALDO), catalase-peroxidase (CAT), lactaldehyde dehydrogenase (ALDH), glycerate 2-kinase (GK), phosphopyruvate hydratase (PPH), phosphoenolpyruvate carboxylase (PPC), L-aspartate dehydrogenase (ASPD), and polyphosphate kinase (PPK), to convert the raw materials into L-Asp in one-pot coupled with NADH and ATP regeneration. Under optimal reaction conditions, 18.6 mM of L-Asp could be produced within 2.0 h at a total enzyme addition of 4.85 mg/mL, demonstrating the high efficiency and productivity characteristics of the designed system. Our technological application provides new insights and methods for the development of biomass resource utilization technologies.

Asunto(s)

Ácido Aspártico , Dióxido de Carbono , Glicerol , Ácido Aspártico/metabolismo , Glicerol/metabolismo , Glicerol/química , Dióxido de Carbono/metabolismo , Biomasa

RESUMEN

Bioresorbable shape memory polymers (SMP) are an emerging class of polymers that can help address several challenges associated with minimally invasive surgery by providing a solution for structural tissue repair. Like most synthetic polymer networks, SMPs require additional biorelevance and modification for biomedical applications. Methodologies used to incorporate bioactive ligands must preserve SMP thermomechanics and ensure biofunctionality following in vivo delivery. We have previously described the development of a novel thermoresponsive bioresorbable SMP, poly (glycerol dodecanedioate) (PGD). In this study, cell-adhesive peptide sequences RGD and YIGSR were conjugated with PGD. We investigated 1) the impact of conjugated peptides on the fixity (Rf), recovery (Rr), and recovery rate (dRr/dT), 2) the impact of conjugated peptides on cell binding, and 3) the impact of the shape memory cycle (Tprog) on conjugated peptide functionality towards binding human bone marrow stromal cells (BMSC). Peptide conjugation conditions impact fixity but not the recovery or recovery rate (p < 0.01). Peptide-conjugated substrates increased cell attachment and proliferation compared with controls (p < 0.001). Using complementary integrin binding cell-adhesive peptides increased proliferation compared with using single peptides (p < 0.05). Peptides bound to PGD substrates exhibited specificity to their respective integrin targets. Following the shape memory cycle, peptides maintained functionality and specificity depending on the shape memory cycle conditions (p < 0.001). The dissipation of strain energy during recovery can drive differential arrangement of conjugated sequences impacting functionality, an important design consideration for functionalized SMPs. STATEMENT OF SIGNIFICANCE: Shape memory elastomers are an emerging class of polymers that are well-suited for minimally invasive repair of soft tissues. Tissue engineering approaches commonly utilize biodegradable scaffolds to deliver instructive cues, including cells and bioactive signals. Delivering these instructive cues on biodegradable shape memory elastomers requires modification with bioactive ligands. Furthermore, it is necessary to ensure the specificity of the ligands to their biological targets when conjugated to the polymer. Moreover, the bioactive ligand functionality must be conserved after completing the shape memory cycle, for applications in tissue engineering.

Asunto(s)

Adhesión Celular , Células Madre Mesenquimatosas , Oligopéptidos , Humanos , Oligopéptidos/química , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Adhesión Celular/efectos de los fármacos , Polímeros/química , Materiales Inteligentes/química , Glicerol/química , Glicerol/análogos & derivados

RESUMEN

Glycerine pitch is a highly alkaline residue from the oleochemical industry that contains glycerol and contaminants, such as water, soap, salt and ash. In this study, acidic heterogeneous glycerol-based carbon catalysts were synthesised for biodiesel production via single-step partial carbonisation and sulfonation using pure glycerol and glycerine pitch, producing products labelled as SGC and SGPC, respectively. Carbon materials were obtained by heating glycerol and concentrated sulfuric acid (1:3) at 200℃ for 1 h. The produced SGC and SGPC displayed high densities of sulfonic group (-SO3H), i.e. 1.49 and 1.00 mmol·g-1, respectively, alongside carboxylic (-COOH) and phenolic (-OH) acid. In the catalytic evaluation, excellent oleic acid conversions of 96.0 ± 0.4 % and 92.4 ± 0.5 % were achieved using SGC and SGPC, respectively, under optimised reaction conditions: 1:10 M ratio of oleic acid to methanol, 5 % (w/w) catalyst, 64℃ and 5 h. SGPC was found to be recyclable with 68.5 % conversion after the 6th cycle, which was attributed to the loss of -SO3H and catalyst deactivation by the deposition of oleic acid on its surface. Remarkably, despite the impurities present in the glycerine pitch, the obtained results demonstrated that the reactivity of SGPC is comparable to SGC and superior to that of commercial solid acid catalysts, which demonstrated that the presence of impurities appears to have minimal impact on the production of carbon materials and their properties.

Asunto(s)

Biocombustibles , Carbono , Glicerol , Glicerol/química , Catálisis , Biocombustibles/análisis , Carbono/química , Ácidos Sulfónicos/química , Ácido Oléico/química , Ácidos Sulfúricos/química , Residuos Industriales

RESUMEN

Viscosity of protein solutions is a critical product quality attribute for protein therapeutics such as monoclonal antibodies. Here we introduce a portable single-use analytical chip-based viscometer for determining the viscosity of protein solutions using low sample volumes of 10 µL. Through the combined use of a microfluidic viscometer, a smartphone camera for image capture, and an automated data processing algorithm for the calculation of the viscosity of fluids, we enable measurement of viscosity of multiple samples in parallel. We first validate the viscometer using glycerol-water mixtures and subsequently demonstrate the ability to perform rapid characterization of viscosity in four different monoclonal antibody formulations in a broad concentration (1 to 320 mg/mL) and viscosity (1 to 600 cP) range, showing excellent agreement with values obtained by a conventional cone-plate rheometer. Not only does the platform offer benefits of viscosity measurements using minimal sample volumes, but enables higher throughput compared to gold-standard methodologies owing to multiplexing of the measurement and single-use characteristics of the viscometer, thus showing great promise in developability studies. Additionally, as our platform has the capability of performing viscosity measurements at the point of sample collection, it offers the opportunity to employ viscosity measurement as an in situ quality control of therapeutic proteins and antibodies.

Asunto(s)

Anticuerpos Monoclonales , Control de Calidad , Viscosidad , Anticuerpos Monoclonales/química , Técnicas Analíticas Microfluídicas/instrumentación , Proteínas/química , Proteínas/análisis , Dispositivos Laboratorio en un Chip , Soluciones , Glicerol/química

RESUMEN

In biomedical and biotechnological domains, liquid protein formulations are vital tools, offering versatility across various fields. However, maintaining protein stability in a liquid form presents challenges due to environmental factors, driving research to refine formulations for broader applications. In our recent study, we investigated the relationship between deep eutectic solvents (DESs) and the natural presence of osmolytes in specific combinations, showcasing the effectiveness of a bioinspired osmolyte-based DES in stabilizing a model protein. Recognizing the need for a more nuanced understanding of osmolyte-based DES stabilization capabilities under different storage conditions, here we broadened the scope of our osmolyte-based DES experimental screening, and delved deeper into structural changes in the enzyme under these conditions. We subjected lysozyme solutions in DESs based on various kosmotropic osmolytes (TMAO, betaine, sarcosine, DMSP, ectoine, GPC, proline, sorbitol and taurine) paired either with another kosmotropic (glycerol) or with chaotropic osmolyte urea to rigorous conditions: heat shock (at 80 °C) and repetitive freeze-thaw cycles (at -20 and -80 °C). Changes in enzyme activity, colloidal stability, and conformational alterations were then monitored using bioassays, aggregation tests, and spectroscopic techniques (FT-IR and CD). Our results demonstrate the remarkable effectiveness of osmolyte-based DES in stabilizing lysozyme under stress conditions, with sarcosine- and betaine-based DESs containing glycerol as a hydrogen bond donor showing the highest efficacy, even at high enzyme loadings up to 200 mg ml-1. Investigation of the individual and combined effects of the DES components on enzyme stability confirmed the synergistic behavior of the kosmotrope-urea mixtures and the cumulative effects in kosmotrope-glycerol mixtures. Additionally, we have shown that the interplay between the enzyme's active and stable (but inactive) states is highly influenced by the water content in DESs. Finally, toxicity assessments of osmolyte-based DESs using cell lines (Caco-2, HaCaT, and HeLa) revealed no risks to human health.

Asunto(s)

Muramidasa , Solventes , Muramidasa/química , Muramidasa/metabolismo , Solventes/química , Humanos , Calor , Glicerol/química , Frío , Estabilidad Proteica , Estabilidad de Enzimas , Animales , Materiales Biocompatibles/química

RESUMEN

Pre-treatment of diamond surface in low-temperature plasma for oxygenation and in acids for carboxylation was hypothesized to promote the branching density of the hyperbranched glycidol polymer. This was expected to increase the homogeneity of the branching level and suppress interactions with proteins. As a result, composite nanodiamonds with reduced hydrodynamic diameters that are maintained in physiological environments were anticipated. Surfaces of 140-nm-sized nanodiamonds were functionalized with oxygen and carboxyl groups for grafting of hyperbranched dendritic polyglycerol via anionic ring-opening polymerization of glycidol. The modification was verified with Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy. Dynamic light scattering investigated colloidal stability in pH-diverse (2-12) solutions, concentrated phosphate-buffered saline, and cell culture media. Thermogravimetric analysis of nanodiamonds-protein incubations examined non-specific binding. Fluorescence emission was tested across pH conditions. Molecular dynamics simulations modeled interparticle interactions in ionic solutions. The hyperbranched polyglycerol grafting increased colloidal stability of nanodiamonds across diverse pH, high ionic media like 10 × concentrated phosphate-buffered saline, and physiological media like serum and cell culture medium. The hyperbranched polyglycerol suppressed non-specific protein adsorption while maintaining intensive fluorescence of nanodiamonds regardless of pH. Molecular modelling indicated reduced interparticle interactions in ionic solutions correlating with the improved colloidal stability.

Asunto(s)

Coloides , Nanodiamantes , Polímeros , Propiedades de Superficie , Nanodiamantes/química , Coloides/química , Concentración de Iones de Hidrógeno , Polímeros/química , Tamaño de la Partícula , Glicerol/química , Dendrímeros/química , Simulación de Dinámica Molecular

RESUMEN

Increased disulfide crosslinking of secreted mucins causes elevated viscoelasticity of mucus and is a key determinant of mucus dysfunction in patients with cystic fibrosis (CF) and other muco-obstructive lung diseases. In this study, we describe the synthesis of a novel thiol-containing, sulfated dendritic polyglycerol (dPGS-SH), designed to chemically reduce these abnormal crosslinks, which we demonstrate with mucolytic activity assays in sputum from patients with CF. This mucolytic polymer, which is based on a reportedly anti-inflammatory polysulfate scaffold, additionally carries multiple thiol groups for mucolytic activity and can be produced on a gram-scale. After a physicochemical compound characterization, we compare the mucolytic activity of dPGS-SH to the clinically approved N-acetylcysteine (NAC) using western blot studies and investigate the effect of dPGS-SH on the viscoelastic properties of sputum samples from CF patients by oscillatory rheology. We show that dPGS-SH is more effective than NAC in reducing multimer intensity of the secreted mucins MUC5B and MUC5AC and demonstrate significant mucolytic activity by rheology. In addition, we provide data for dPGS-SH demonstrating a high compound stability, low cytotoxicity, and superior reaction kinetics over NAC at different pH levels. Our data support further development of the novel reducing polymer system dPGS-SH as a potential mucolytic to improve mucus function and clearance in patients with CF as well as other muco-obstructive lung diseases.

Asunto(s)

Glicerol , Polímeros , Esputo , Compuestos de Sulfhidrilo , Humanos , Glicerol/química , Polímeros/química , Polímeros/farmacología , Esputo/metabolismo , Esputo/química , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacología , Fibrosis Quística/metabolismo , Fibrosis Quística/tratamiento farmacológico , Mucina 5AC/metabolismo , Enfermedades Pulmonares Obstructivas/tratamiento farmacológico , Enfermedades Pulmonares Obstructivas/metabolismo , Mucina 5B/metabolismo , Sulfatos/química , Sulfatos/farmacología , Expectorantes/farmacología , Expectorantes/química , Moco/metabolismo , Moco/química , Reología , Acetilcisteína/farmacología , Acetilcisteína/química , Viscosidad

RESUMEN

Lignin represents a promising source of renewable energy. The development of CLEO (Cold processed Lignin Ethanol Oil) fuel introduces a novel lignin valorization approach, proposing its potential as maritime biofuel. However, its industrial success depends on enhancing fractionation yields and reducing solvent evaporation, which necessitates a detailed analysis of lignin properties, solvent types, and process parameters. By using novel combinations of biobased solvents, yields improved from 34 wt% to 49-53 wt% by using 30 wt% water or 40 wt% glycerol in ethanol, where Hildebrand Solubility Parameters emerged as indicative tool for increasing yields. Experiments on solid-to-liquid (S:L) ratios revealed a good balance between yield and lignin dispersion concentration at an S:L of 1:2.5. Producing CLEO with an improved solvent composition and S:L ratio resulted in 89 wt% yield while eliminating solvent evaporation requirements. This study highlights the potential for enhancing CLEO production efficiency and advancing it to industrial scale.

Asunto(s)

Biocombustibles , Etanol , Lignina , Solventes , Temperatura , Lignina/química , Solventes/química , Etanol/química , Solubilidad , Glicerol/química , Alcoholes/química , Biotecnología/métodos

RESUMEN

1H-1H nuclear cross-relaxation experiments have been carried out with lysozyme in variable glycerol viscosity to study intramolecular motion, self-diffusion, and isotropic rigid-body rotational tumbling at 298 K, pH 3.8. Dynamics of intramolecular 1H-1H cross-relaxation rates, the increase in internuclear spatial distances, and lateral and rotational diffusion coefficients all show fractional viscosity dependence with a power law exponent κ in the 0.17-0.83 range. The diffusion coefficient of glycerol Ds with the bulk viscosity itself is non-Stokesian, having a fractional viscosity dependence on the medium viscosity (Ds âˆ¼ Î·-κ, κ ≈ 0.71). The concurrence and close similarity of the fractional viscosity dependence of glycerol diffusion on the one hand, and diffusion and intramolecular cross-relaxation rates of the protein on the other lead to infer that relaxation of glycerol slaves protein relaxations. Glycerol-transformed native lysozyme to a quasi-native state does not affect the conclusion that both global and internal fluctuations are slaved to glycerol relaxation.

Asunto(s)

Glicerol , Muramidasa , Muramidasa/química , Muramidasa/metabolismo , Glicerol/química , Viscosidad , Espectroscopía de Protones por Resonancia Magnética , Resonancia Magnética Nuclear Biomolecular , Difusión , Animales , Pollos

RESUMEN

Cryoprotective agents play a critical role in minimizing cell damage caused by ice formation during cryopreservation. However, high concentrations of CPAs are toxic to cells and tissues. Required concentrations of CPAs can be reduced by utilizing higher cooling and warming rates, but insight into the thermophysical properties of biological solutions in the vitrification method is necessary for the development of cryopreservation protocols. Most studies on thermophysical properties under ultra-rapid cooling conditions have been qualitatively based on visualization. Differential scanning calorimetry methods are ideal for studying the behavior of biomaterials in various freezing conditions quantitatively and accurately, though previous studies have been predominantly restricted to slower cooling rates. Here, we developed an ultra-rapid cooling method for DSC that can achieve minimal cooling rates exceeding 2000 °C/min. We investigated the thermophysical vitrification behavior of ternary solutions of phosphate buffer saline (1X), dimethyl sulfoxide or glycerol and ice blocking polymers (X-1000 or Z-1000). We quantified the impact of solute concentration on ice crystal formation during rapid cooling. Our findings support the expectation that increasing the solute concentration reduces the amount of ice formation, including devitrification. Devitrification increases from 0 % to 40 % (v/v) Me2SO and then reduces significantly. The relative amounts of devitrification to the total ice formation are 0 %, 60 %, 0 % in 20 %, 40 %, 60 % (v/v) Me2SO, and 2 %, 48 %, 49 % in 20 %, 40 %, 60 % (v/v) glycerol, respectively. The results suggest that at low concentrations, such as below 20 % (v/v) for Me2SO or glycerol, increasing the warming rate after ultra-rapid freezing is not essential to eliminate devitrification. Furthermore, ice blocking polymers do not reduce ice formation substantially and cannot eliminate devitrification under ultra-rapid cooling conditions. In conclusion, our results provide insights into the impact of solute concentration on ice formation and devitrification during rapid cooling, which can be practical for optimizing cryopreservation protocols.

Asunto(s)

Rastreo Diferencial de Calorimetría , Criopreservación , Crioprotectores , Dimetilsulfóxido , Glicerol , Vitrificación , Criopreservación/métodos , Crioprotectores/química , Crioprotectores/farmacología , Dimetilsulfóxido/química , Glicerol/química , Glicerol/farmacología , Congelación , Hielo

RESUMEN

Microbial strain improvement through adaptive laboratory evolution (ALE) has been a key strategy in biotechnology for enhancing desired phenotypic traits. In this Biotech Method paper, we present an accelerated ALE (aALE) workflow and its successful implementation in evolving Cupriavidus necator H16 for enhanced tolerance toward elevated glycerol concentrations. The method involves the deliberate induction of genetic diversity through controlled exposure to divalent metal cations, enabling the rapid identification of improved variants. Through this approach, we observed the emergence of robust variants capable of growing in high glycerol concentration environments, demonstrating the efficacy of our aALE workflow. When cultivated in 10% v/v glycerol, the adapted variant Mn-C2-B11, selected through aALE, achieved a final OD600 value of 56.0 and a dry cell weight of 15.2 g L-1, compared to the wild type (WT) strain's final OD600 of 39.1 and dry cell weight of 8.4 g L-1. At an even higher glycerol concentration of 15% v/v, Mn-C2-B11 reached a final OD600 of 48.9 and a dry cell weight of 12.7 g L-1, in contrast to the WT strain's final OD600 of 9.0 and dry cell weight of 3.1 g L-1. Higher glycerol consumption by Mn-C2-B11 was also confirmed by high-performance liquid chromatography (HPLC) analysis. This adapted variant consumed 34.5 times more glycerol compared to the WT strain at 10% v/v glycerol. Our method offers several advantages over other reported ALE approaches, including its independence from genetically modified strains, specialized genetic tools, and potentially carcinogenic DNA-modifying agents. By utilizing divalent metal cations as mutagens, we offer a safer, more efficient, and cost-effective alternative for expansion of genetic diversity. With its ability to foster rapid microbial evolution, aALE serves as a valuable addition to the ALE toolbox, holding significant promise for the advancement of microbial strain engineering and bioprocess optimization.

Asunto(s)

Cupriavidus necator , Glicerol , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Glicerol/metabolismo , Glicerol/química , Cationes Bivalentes , Evolución Molecular Dirigida/métodos