Spin-Induced Organization of Cellulose Nanocrystals.

Cellulose nanocrystals (CNCs) are composed of chiral cellulose units, which form chiral nematic liquid crystals in water that, upon drying, self-assemble to more complex spiral chiral sheets. This secondary structure arrangement is found to change with an external magnetic or electric field. Here, we show that one of the basic organization driving forces is electron spin, which is produced as the charge redistributes in the organization process of the chiral building blocks. It is important to stress that the electron spin-exchange interactions supply the original driving force and not the magnetic field per se. The results present the first utilization of the chiral-induced spin selectivity (CISS) effect in sugars, enabling one to regulate the CNC bottom-up fabrication process. Control is demonstrated on the organization order of the CNC by utilizing different magnetization directions of the ferromagnetic surface. The produced spin is probed using a simple Hall device. The measured Hall resistance shows that the CNC sheets' arrangement is affected during the first four hours as long as the CNC is in its wet phase. On introducing the 1,2,3,4-butanetetracarboxylic acid cross-linker into the CNC sheet, the packing density of the CNC helical structure is enhanced, presenting an increase in the Hall resistance and the chiral state.

Foliar Application of dsRNA Targeting Endogenous Potato (Solanum tuberosum) Isoamylase Genes ISA1, ISA2, and ISA3 Confers Transgenic Phenotype.

Isoamylase (ISA) is a debranching enzyme found in many plants, which hydrolyzes (1-6)-α-D glucosidic linkages in starch, amylopectin, and ß-dextrins, and is thought to be responsible for starch granule formation (ISA1 and ISA2) and degradation (ISA3). Lipid-modified PEI (lmPEI) was synthesized as a carrier for long double-stranded RNA (dsRNA, 250-bp), which targets the three isoamylase isoforms. The particles were applied to the plant via the foliar spray and were differentially effective in suppressing the expressions of ISA1 and ISA2 in the potato leaves, and ISA3 in the tubers. Plant growth was not significantly impaired, and starch levels in the tubers were not affected as well. Interestingly, the treated plants had significantly smaller starch granule sizes as well as increased sucrose content, which led to an early sprouting phenotype. We confirm the proposal of previous research that an increased number of small starch granules could be responsible for an accelerated turnover of glucan chains and, thus, the rapid synthesis of sucrose, and we propose a new relationship between ISA3 and the starch granule size. The implications of this study are in achieving a transgenic phenotype for endogenous plant genes using a systemic, novel delivery system, and foliar applications of dsRNA for agriculture.

Foliar Delivery of siRNA Particles for Treating Viral Infections in Agricultural Grapevines.

Grapevine leafroll disease (GLD) is a globally spreading viral infection that causes major economic losses by reducing crop yield, plant longevity and berry quality, with no effective treatment. Grapevine leafroll associated virus-3 (GLRaV-3) is the most severe and prevalent GLD strain. Here, we evaluated the ability of RNA interference (RNAi), a non-GMO gene-silencing pathway, to treat GLRaV-3 in infected Cabernet Sauvignon grapevines. We synthesized lipid-modified polyethylenimine (lmPEI) as a carrier for long double-stranded RNA (dsRNA, 250-bp-long) that targets RNA polymerase and coat protein genes that are conserved in the GLRaV-3 genome. Self-assembled dsRNA-lmPEI particles, 220 nm in diameter, displayed inner ordered domains spaced 7.3±2 nm from one another, correlating to lmPEI wrapping spirally around the dsRNA. The particles effectively protected RNA from degradation by ribonucleases, and Europium-loaded particles applied to grapevine leaves were detected as far as 60-cm from the foliar application point. In three field experiments, a single dose of foliar administration knocked down GLRaV-3 titer, and multiple doses of the treatment kept the viral titer at baseline and triggered recovery of the vine and berries. This study demonstrates RNAi as a promising platform for treating viral diseases in agriculture.

3D Printing of Cellulose Nanocrystal-Loaded Hydrogels through Rapid Fixation by Photopolymerization.

New ink compositions for direct ink writing (DIW) printing of hydrogels, combining superior rheological properties of cellulose nanocrystals (CNCs) and a water-compatible photoinitiator, are presented. Rapid fixation was achieved by photopolymerization induced immediately after the printing of each layer by 365 nm light for 5 s, which overcame the common height limitation in DIW printing of hydrogels, and enabled the fabrication of objects with a high aspect ratio. CNCs imparted a unique rheological behavior, which was expressed by orders of magnitude difference in viscosity between low and high shear rates and in rapid high shear recovery, without compromising ink printability. Compared to the literature, the presented printing compositions enable the use of low photoinitiator concentrations at a very short build time, 6.25 s/mm, and are also curable by 405 nm light, which is favorable for maintaining viability in bioinks.

BactoSpin: Novel Technology for Rapid Bacteria Detection and Antibiotic Susceptibility Testing.

Inappropriate use of antibiotics is one of the leading causes of the increasing numbers of resistant bacteria strains, resulting in 700,000 deaths worldwide each year. Reducing unnecessary use of antibiotics and choosing the most effective antibiotics instead of broad-spectrum drugs will slow the arms race between germs and humans. Urinary tract infections (UTIs) are among the most common bacterial infections. Currently, accurate diagnosis of UTI requires approximately 48 h from the time of urine sample collection until antibiotic susceptibility test (AST) results. This work presents a rapid bacterial detection device that integrates a centrifuge, microscope, and incubator. Two disposable microfluidic chips were developed. The first chip was designed for bacteria concentration, detection, and medium exchange. A second multi-channel chip was developed for AST. This chip contains superhydrophobic and hydrophilic coatings to ensure liquid separation between the channels without the need for valves. The designed chips supported the detection of E. coli at a concentration as low as 5 × 103 cells/mL within 5 min and AST in under 2 h. AST was also successfully performed with Klebsiella pneumonia isolated from a human urine sample. In addition, machine-learning-based image recognition was shown to reduce the required time for AST and to provide results within 1 h for E. coli cells. Thus, the BactoSpin device can serve as an efficient and rapid platform for UTI diagnostics and AST.

A Paper-Based Near-Infrared Optical Biosensor for Quantitative Detection of Protease Activity Using Peptide-Encapsulated SWCNTs.

A protease is an enzyme that catalyzes proteolysis of proteins into smaller polypeptides or single amino acids. As crucial elements in many biological processes, proteases have been shown to be informative biomarkers for several pathological conditions in humans, animals, and plants. Therefore, fast, reliable, and cost-effective protease biosensors suitable for point-of-care (POC) sensing may aid in diagnostics, treatment, and drug discovery for various diseases. This work presents an affordable and simple paper-based dipstick biosensor that utilizes peptide-encapsulated single-wall carbon nanotubes (SWCNTs) for protease detection. Upon enzymatic digestion of the peptide, a significant drop in the photoluminescence (PL) of the SWCNTs was detected. As the emitted PL is in the near-infrared region, the developed biosensor has a good signal to noise ratio in biological fluids. One of the diseases associated with abnormal protease activity is pancreatitis. In acute pancreatitis, trypsin concentration could reach up to 84 µg/mL in the urine. For proof of concept, we demonstrate the feasibility of the proposed biosensor for the detection of the abnormal levels of trypsin activity in urine samples.

Surface Charge Influence on the Phase Separation and Viscosity of Cellulose Nanocrystals.

A series of four cellulose nanocrystal (CNC) suspensions were prepared from bleached softwood kraft pulp using different conditions of sulfuric acid hydrolysis. The CNCs were identical in size (95 nm in length × 5 nm in width) but had different surface charges corresponding to the harshness of the hydrolysis conditions. Consequently, it was possible to isolate the effects of surface charge on the self-assembly and viscosity of the CNC suspensions across surface charges ranging from 0.27%S to 0.89%S. The four suspensions (never-dried, free of added electrolyte) all underwent liquid crystalline phase separation, but the concentration onset for the emergence of the chiral nematic phase shifted to higher values with increasing surface charge. Similarly, suspension viscosity was also influenced by surface charge, with suspensions of lower surface charge CNCs more viscous and tending to gel at lower concentrations. The properties of the suspensions were interpreted in terms of the increase in effective diameter of the nanocrystals due to the surface electrostatic repulsion of the negative sulfate half-esters, as modified by the screening effects of the H+ counterions in the suspensions. The results suggest that there is a threshold surface charge density (∼0.3%S) above which effective volume considerations are dominant across the concentration range relevant to liquid crystalline phase formation. Above this threshold value, phase separation occurs at the same effective volume fraction of CNCs (∼10 vol %), with a corresponding increase in critical concentration due to the decrease in effective diameter that occurs with increasing surface charge. Below or near this threshold value, the formation of end-to-end aggregates may favor gelation and interfere with ordered phase formation.

Identification of genes related to skin development in potato.

KEY MESSAGE: Newly identified genes that are preferentially expressed in potato skin include genes that are associated with the secondary cell wall and stress-related activities and contribute to the skin's protective function. Microarrays were used to compare the skin and tuber-flesh transcriptomes of potato, to identify genes that contribute to the unique characteristics of the skin as a protective tissue. Functional gene analysis indicated that genes involved in developmental processes such as cell division, cell differentiation, morphogenesis and secondary cell wall formation (lignification and suberization), and stress-related activities, are more highly expressed in the skin than in the tuber flesh. Several genes that were differentially expressed in the skin (as verified by qPCR) and had not been previously identified in potato were selected for further analysis. These included the StKCS20-like, StFAR3, StCYP86A22 and StPOD72-like genes, whose sequences suggest that they may be closely related to known suberin-related genes; the StHAP3 transcription factor that directs meristem-specific expression; and the StCASP1B2-like and StCASP1-like genes, which are two orthologs of a protein family that mediates the formation of Casparian strips in the suberized endodermis of Arabidopsis roots. An examination of microtubers induced from transgenic plants carrying GUS reporter constructs of these genes indicated that these genes were expressed in the skin, with little to no expression in the tuber flesh. Some of the reporter constructs were preferentially expressed in the inner layers of the skin, the root endodermis, the vascular cambium and the epidermis of the stem. Cis-regulatory elements within the respective promoter sequences support this gene-expression pattern.

Spider Silk-CBD-Cellulose Nanocrystal Composites: Mechanism of Assembly.

The fabrication of cellulose-spider silk bio-nanocomposites comprised of cellulose nanocrystals (CNCs) and recombinant spider silk protein fused to a cellulose binding domain (CBD) is described. Silk-CBD successfully binds cellulose, and unlike recombinant silk alone, silk-CBD self-assembles into microfibrils even in the absence of CNCs. Silk-CBD-CNC composite sponges and films show changes in internal structure and CNC alignment related to the addition of silk-CBD. The silk-CBD sponges exhibit improved thermal and structural characteristics in comparison to control recombinant spider silk sponges. The glass transition temperature (Tg) of the silk-CBD sponge was higher than the control silk sponge and similar to native dragline spider silk fibers. Gel filtration analysis, dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (TEM) indicated that silk-CBD, but not the recombinant silk control, formed a nematic liquid crystalline phase similar to that observed in native spider silk during the silk spinning process. Silk-CBD microfibrils spontaneously formed in solution upon ultrasonication. We suggest a model for silk-CBD assembly that implicates CBD in the central role of driving the dimerization of spider silk monomers, a process essential to the molecular assembly of spider-silk nanofibers and silk-CNC composites.

Construction of a highly stable artificial glutathione peroxidase on a protein nanoring.

Stable Protein One (SP1) is a boiling-stable oligomeric protein. The unique characteristics of SP1 offer a scaffold to design artificial enzymes against extreme temperature. Here, an efficient antioxidase is successfully constructed on the ring-shaped SP1 homododecamer. By means of computational design and genetic engineering, the active center of glutathione peroxidase (GPx), selenocysteine (Sec), is introduced to the SP1 monomer surface, and the self-assembly properties of the protein monomer lead to a ring-shaped SP1 with homododecamer catalytic selenium centers. This artificial selenoenzyme exhibits high GPx catalytic activity and shows a typical ping-pong kinetic mechanism. Moreover, it has a significantly broader temperature range and high thermostability. Owing to having multi-GPx active centers on a SP1 oligomer, this selenium-containing biomacromolecule exerts an excellent capability to protect cells from oxidative damage at the mitochondrial level. This strategy represents a new way to develop thermostable artificial nanoenzymes for some specific applications.

Cultivation of Bovine Mesenchymal Stem Cells on Plant-Based Scaffolds in a Macrofluidic Single-Use Bioreactor for Cultured Meat.

Reducing production costs, known as scaling, is a significant obstacle in the advancement of cultivated meat. The cultivation process hinges on several key components, e.g., cells, media, scaffolds, and bioreactors. This study demonstrates an innovative approach, departing from traditional stainless steel or glass bioreactors, by integrating food-grade plant-based scaffolds and thermoplastic film bioreactors. While thermoplastic films are commonly used for constructing fluidic systems, conventional welding methods are cost-prohibitive and lack rapid prototyping capabilities, thus inflating research and development expenses. The developed laser welding technique facilitates contamination-free and leakproof sealing of polyethylene films, enabling the efficient fabrication of macrofluidic systems with various designs and dimensions. By incorporating food-grade plant-based scaffolds, such as rice seeded with bovine mesenchymal stem cells, into these bioreactors, this study demonstrates sterile cell proliferation on scaffolds within macrofluidic systems. This approach not only reduces bioreactor prototyping and construction costs but also addresses the need for scalable solutions in both research and industrial settings. Integrating single-use bioreactors with minimal shear forces and incorporating macro carriers such as puffed rice may further enhance biomass production in a scaled-out model. The use of food-grade plant-based scaffolds aligns with sustainable practices in tissue engineering and cultured-meat production, emphasizing its suitability for diverse applications.

Oxidized cellulose binding to allergens with a carbohydrate-binding module attenuates allergic reactions.

Grass and mite allergens are of the main causes of allergy and asthma. A carbohydrate-binding module (CBM) represents a common motif to groups I (ß-expansin) and II/III (expansin-like) grass allergens and is suggested to mediate allergen-IgE binding. House dust mite group II allergen (Der p 2 and Der f 2) structures bear strong similarity to expansin's CBM, suggesting their ability to bind carbohydrates. Thus, this study proposes the design of a carbohydrate-based treatment in which allergen binding to carbohydrate particles will promote allergen airway clearance and prevent allergic reactions. The aim of the study was to identify a polysaccharide with high allergen-binding capacities and to explore its ability to prevent allergy. Oxidized cellulose (OC) demonstrated allergen-binding capacities toward grass and mite allergens that surpassed those of any other polysaccharide examined in this study. Furthermore, inhalant preparations of OC microparticles attenuated allergic lung inflammation in rye grass-sensitized Brown Norway rats and OVA-sensitized BALB/c mice. Fluorescently labeled OC efficiently cleared from the mouse airways and body organs. Moreover, long-term administration of OC inhalant to Wistar rats did not result in toxicity. In conclusion, many allergens, such as grass and dust mite, contain a common CBM motif. OC demonstrates a strong and relatively specific allergen-binding capacity to CBM-containing allergens. OC's ability to attenuate allergic inflammation, together with its documented safety record, forms a firm basis for its application as an alternative treatment for prevention and relief of allergy and asthma.

Biocompatible, Resilient, and Tough Nanocellulose Tunable Hydrogels.

Hydrogels have been proposed as potential candidates for many different applications. However, many hydrogels exhibit poor mechanical properties, which limit their applications. Recently, various cellulose-derived nanomaterials have emerged as attractive candidates for nanocomposite-reinforcing agents due to their biocompatibility, abundance, and ease of chemical modification. Due to abundant hydroxyl groups throughout the cellulose chain, the grafting of acryl monomers onto the cellulose backbone by employing oxidizers such as cerium(IV) ammonium nitrate ([NH4]2[Ce(NO3)6], CAN) has proven a versatile and effective method. Moreover, acrylic monomers such as acrylamide (AM) may also polymerize by radical methods. In this work, cerium-initiated graft polymerization was applied to cellulose-derived nanomaterials, namely cellulose nanocrystals (CNC) and cellulose nanofibrils (CNF), in a polyacrylamide (PAAM) matrix to fabricate hydrogels that display high resilience (~92%), high tensile strength (~0.5 MPa), and toughness (~1.9 MJ/m3). We propose that by introducing mixtures of differing ratios of CNC and CNF, the composite's physical behavior can be fine-tuned across a wide range of mechanical and rheological properties. Moreover, the samples proved to be biocompatible when seeded with green fluorescent protein (GFP)-transfected mouse fibroblasts (3T3s), showing a significant increase in cell viability and proliferation compared to samples comprised of acrylamide alone.

In Situ Synthesis of Keratin and Melanin Chromophoric Submicron Particles.

In humans, melanin plays an esthetic role, dictating hair and skin color and traits, while keratin is the protein that comprises most of the epidermis layer. Eumelanin and pheomelanin are types of melanin synthesized from the same building blocks via enzymatic oxidation. Pheomelanin has an additional building block, cysteine amino acid, which affects its final structure. Keratin contains high cysteine content, and by exploiting free thiols in hydrolyzed keratin, we demonstrate the formation of keratin-melanin (KerMel) chromophoric submicron particles. Cryo-TEM analyses found KerMel particle sizes to be 100-300 nm and arranged in the form of a central keratin particle with polymerized l-dopa chains. Attenuated total reflection (ATR)-FTIR, UV-vis, and fluorescence measurements identified new chemical bonds, indicating the formation of KerMel particles. Finally, KerMel replicated natural skin tones and proved cytocompatibility for human epidermal keratinocytes at concentrations below 0.1 mg/mL. Taken together, KerMel is a novel, tunable material that has the potential to integrate into the cosmetic industry.

Biobased Electronics: Tunable Dielectric and Piezoelectric Cellulose Nanocrystal-Protein Films.

Cellulose has been a go-to material for its dielectric properties from the onset of capacitor development. The demand for an energy storage solution continues to grow, but the supply remains limited and relies too often on fossil and mined materials. This work proposes a fully sustainable and green method with which to produce dielectric thin films made of renewable and degradable materials. Cellulose nanocrystals (CNC) made an excellent matrix for the dispersion of proteins and the fabrication of robust transparent thin films with enhanced dielectric permittivity. A range of proteins sources, additives and concentrations allowed for us to control the dielectric permittivity from εr = 4 to 50. The proteins screened came from animal and plant sources. The films were formed from drying a water suspension of the CNC and proteins through evaporation-induced self-assembly. This yielded nano-layered structures with very high specific surface areas, ideal for energy storage devices. The resulting films were characterized with respect to the electrical, mechanical, piezoelectric, and optical properties to be compared. Electrically conductive (σ = 1.53 × 103 S/m) CNC films were prepared with carbon nanotubes (CNT). The fabricated films were used to make flexible, sustainable, and degradable capacitors by layering protein-based films between CNC-CNT composite films.

Mosquito bite prevention through self-assembled cellulose nanocrystals.

Mosquitoes are the deadliest of all combined insects and animals affecting millions and killing hundreds or thousands of people each year. Existing protection methods however are limited and include volatile compounds that actively repel mosquitoes such as N,N-Diethyl-meta-toluamide (DEET) or different essential oils such as geraniol and citronella. Most are odorous compounds and require organic solvents for dispersion. This work investigates the barrier properties of cellulose nanocrystals (CNCs). CNCs are known to self-assemble in strong, transparent, chemical barrier films. They are fully bio-based, and their surface chemistry is ideal for aqueous dispersion of many compounds. This work saw a significant 80% decrease in feeding on human skin when a thin CNC coat was applied. The effect was further confirmed by artificial feeding on Aedes aegypti wherein CNC appears to act as a chemical camouflage to the many cues sought by the insects. The combined effect of CNC with indole reduced egg laying post exposure to mammalian blood close to null with 99.4% less eggs as compared to control. The chemical barrier effect was assessed through a simple headspace experiment showing that the same CNC coat blocked the passage of ammonium hydroxide vapor, a commonly used mosquito attractant, when applied on a filter paper membrane.

Biochar applications in microbial fermentation processes for producing non-methane products: Current status and future prospects.

The objective of this review is to encourage the technical development of biochar-assisted microbial fermentation. To this end, recent advances in biochar applications for microbial fermentation processes (i.e., non-methane products of hydrogen, acids, alcohols, and biofertilizer) have been critically reviewed, including process performance, enhanced mechanisms, and current research gaps. Key findings of enhanced mechanisms by biochar applications in biochemical conversion platforms are summarized, including supportive microbial habitats due to the immobilization effect, pH buffering due to alkalinity, nutrition supply due to being rich in nutrient elements, promoting electron transfer by acting as electron carriers, and detoxification of inhibitors due to high adsorption capacity. The current technical limitations and biochar's industrial applications in microbial fermentation processes are also discussed. Finally, suggestions like exploring functionalized biochar materials, biochar's automatic addition and pilot-scale demonstration are proposed. This review would further promote biochar applications in microbial fermentation processes for the production of non-methane products.

Formation of hydrophilic nanochannels in the membrane of living cells by the ringlike stable protein-SP1.

The assembly of functional junction between nerve cells and electronic sensing pads is a critical problem in the construction of effective neuroelectronic hybrid systems. Here, we demonstrate for the first time that the ringlike Stable Protein 1 (Sp1) and its derivatives can be used to generate hydrophilic nanochannels in the plasma membrane of living cells. Since SP1-derivatives can be linked to both the plasma membrane, gold or silicon surfaces, they may serve to ohmically link between cells interior and electronic sensing devices.

Wood Warping Composite by 3D Printing.

Wood warping is a phenomenon known as a deformation in wood that occurs when changes in moisture content cause an unevenly volumetric change due to fiber orientation. Here we present an investigation of wood warped objects that were fabricated by 3D printing. Similar to natural wood warping, water evaporation causes volume decrease of the printed object, but in contrast, the printing pathway pattern and flow rate dictate the direction of the alignment and its intensity, all of which can be predesigned and affect the resulting structure after drying. The fabrication of the objects was performed by an extrusion-based 3D printing technique that enables the deposition of water-based inks into 3D objects. The printing ink was composed of 100% wood-based materials, wood flour, and plant-extracted natural binders cellulose nanocrystals, and xyloglucan, without the need for any additional synthetic resins. Two archetypal structures were printed: cylindrical structure and helices. In the former, we identified a new length scale that gauges the effect of gravity on the shape. In the latter, the structure exhibited a shape transition analogous to the opening of a seedpod, quantitatively reproducing theoretical predictions. Together, by carefully tuning the flow rate and printing pathway, the morphology of the fully dried wooden objects can be controlled. Hence, it is possible to design the printing of wet objects that will form different final 3D structures.