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.

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.

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.

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.

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.

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.

One-step double network hydrogels of photocurable monomers and bacterial cellulose fibers.

Soft-tissue replacements are challenging due to the stringent compliance requirements for the implanted materials in terms of biocompatibility, durability, high wear resistance, low friction, and water content. Acrylate hydrogels are worth considering as soft tissue implants as they can be photocurable and sustain customized shapes through 3D bioprinting. However, acrylate-based hydrogels present weak mechanical properties and significant dimensional changes when immersed in liquids. To address these obstacles, we fabricated double network (DN) hydrogels composed of polyacrylic acid (PAA) and bacterial cellulose nanofibers (BCNFs) by one fast UV photopolymerization step. BCNFs/PAA hydrogels with a 0.5 wt% BCNFs content present an increased stiffness and a lower, non-pH-dependent swelling than PAA hydrogels or PAA hydrogels with cellulose nanocrystals. Besides, BCNFs/PAA hydrogels are biocompatible and can be frozen/thawed. Those characteristics endorse these hybrid hydrogels as potential candidates for vascular and cartilage tissue implants.

Ecogeographic Conditions Dramatically Affect Trans-Resveratrol and Other Major Phenolics' Levels in Wine at a Semi-Arid Area.

Grapevines are susceptible and responsive to their surrounding environment. Factors such as climate region and terroir are known to affect polyphenolic compounds in wine and therefore, its quality. The uniqueness of the terroir in Israel is the variety of soil types and the climatic conditions, ranging from Mediterranean to arid climates. Thus, understanding the effects of climate on grapevine performance in Israel may be a test case for the effect of climate change on grapevine at other areas in the future. First, we present a preliminary survey (2012-2014) in different climate zones and terroirs, which showed that trans-resveratrol concentrations in Merlot and Shiraz were high, while those of Cabernet Sauvignon were significantly lower. A further comprehensive countrywide survey (2016) of Merlot wines from 62 vineyards (53 wineries) compared several phenolic compounds' concentrations between five areas of different climate and terroir. Results show a connection between trans-resveratrol concentrations, variety, and terroir properties. Furthermore, we show that trans-resveratrol concentrations are strongly correlated to humidity levels at springtime, precipitation, and soil permeability. This work can be considered a glimpse into the possible alterations of wine composition in currently moderate-climate wine-growing areas.

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.

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.

Poly(vinyl alcohol)-methacrylate with CRGD peptide: A photocurable biocompatible hydrogel.

Polyvinyl alcohol (PVA)-based hydrogels are promising biomaterials for tissue engineering printing applications. However, one of their main disadvantages is their inability to support cell attachment, which is a critical feature for the preparation of biological scaffolds. The goal of this study was to develop a printable, cell-supportive PVA-based bioink with tunable mechanical properties, without using animal-derived polymers which potentially harbor human pathogens. An ultraviolet light (UV) curable PVA-methacrylate (PVA-MA) polymer mixed with Cys-Arg-Gly-Asp (CRGD) peptide was developed. This peptide holds the integrin receptor binding sequence - RGD, that can enhance cell attachment. The additional cysteine was designed to enable its thiol binding under UV to methacrylate groups of the UV curable PVA-MA. Vero cell, as an adherent cell model was used to assess the hydrogel's cell adhesion. It was found that the PVA-MA-CRGD formula enables the preparation of hydrogels with excellent cell attachment and had even shown superior cell attachment properties relative to added gelatin. Adding hyaluronic acid (HA) as a rheologic modulator enabled the printing of this new formula. Our overall data demonstrates the applicability of this mixture as a bioink for soft tissue engineering such as skin, adipose, liver or kidney tissue.

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.

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.

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.

Cellulose Nanocrystals and Corn Zein Oxygen and Water Vapor Barrier Biocomposite Films.

Cellulose nanocrystals (CNC) are well-suited to the preparation of biocomposite films and packaging material due to its abundance, renewability, biodegradability, and favorable film-forming capacity. In this study, different CNC and corn zein (CZ) composite films were prepared by adding CZ to the CNC suspension prior to drying, in order to change internal structure of resulting films. Films were developed to examine their performance as an alternative water vapor and oxygen-barrier for flexible packaging industry. Water vapor permeability (WVP) and oxygen transmission rate (OTR) of the biocomposite films decreased significantly in a specific ratio between CNC and CZ combined with 1,2,3,4-butane tetracarboxylic acid (BTCA), a nontoxic cross linker. In addition to the improved barrier properties, the incorporation of CZ benefitted the flexibility and thermal stability of the CNC/CZ composite films. The toughness increased by 358%, and Young's modulus decreased by 32% compared with the pristine CNC film. The maximum degradation temperature increased by 26 °C, compared with that of CNC film. These results can be attributed to the incorporation of a hydrophobic protein into the matrix creating hydrophobic interactions among the biocomposite components. SEM and AFM analysis indicated that CZ could significantly affect the CNC arrangement, and the film surface topography, due to the mechanical bundling and physical adsorption effect of CZ to CNC. The presented results indicate that CNC/CZ biocomposite films may find applications in packaging, and in multi-functionalization materials.

Regeneration of grade 3 ankle sprain, using the recombinant human amelogenin protein (rHAM+ ) in a rat model.

Lateral ligament tears, also known as high-grade ankle sprains, are common, debilitating, and usually heal slowly. Ten to thirty percent of patients continue to suffer from chronic pain and ankle instability even after 3 to 9 months. Previously, we showed that the recombinant human amelogenin (rHAM+ ) induced regeneration of fully transected rat medial collateral ligament, a common proof-of-concept model. Our aim was to evaluate whether rHAM+ can regenerate torn ankle calcaneofibular ligament (CFL), an important component of the lateral ankle stabilizers. Right CFLs of Sabra rats were transected and treated with 0, 0.5, or 1 µg/µL rHAM+ dissolved in propylene glycol alginate (PGA). Results were compared with the normal group, without surgery. Healing was evaluated 12 weeks after treatment by mechanical testing (ratio between the right and left, untransected ligaments of the same rat), and histology including immunohistochemical staining of collagen I and S100. The mechanical properties, structure, and composition of transected ligaments treated with 0.5 µg/µL rHAM+ (experimental) were similar to untransected ligaments. PGA (control) treated ligaments were much weaker, lax, and unorganized compared with untransected ligaments. Treatment with 1 µg/µL rHAM+ was not as efficient as 0.5 µg/µL rHAM+ . Normal arrangement of collagen I fibers and of proprioceptive nerve endings, parallel to the direction of the force, was detected in ligaments treated with 0.5 µg/µL rHAM+ , and scattered arrangement, resembling scar tissue, in control ligaments. In conclusion, we showed that rHAM+ induced significant mechanical and structural regeneration of torn rat CFLs, which might be translated into treatment for grades 2 and 3 ankle sprain injuries.

A nanoscale paper-based near-infrared optical nose (NIRON).

Electronic noses (e-nose) and optical noses (o-nose) are two emerging approaches for the development of artificial olfactory systems for flavor and smell evaluation. The current work leverages the unique optical properties of semiconducting single-wall carbon nanotubes (SWCNTs) to develop a prototype of a novel paper-based near-infrared optical nose (NIRON). We have drop-dried an array of SWCNTs encapsulated with a wide variety of peptides on a paper substrate and continuously imaged the emitted SWCNTs fluorescence using a CMOS camera. Odors and different volatile molecules were passed above the array in a flow chamber, resulting in unique modulation patterns of the SWCNT photoluminescence (PL). Quartz crystal microbalance (QCM) measurements performed in parallel confirmed the direct binding between the vapor molecules and the peptide-SWCNTs. PL levels measured before and during exposure demonstrate distinct responses to the four tested alcoholic vapors (ethanol, methanol, propanol, and isopropanol). In addition, machine learning tools directly applied to the fluorescence images allow us to distinguish between the aromas of red wine, beer, and vodka. Further, we show that the developed sensor can detect limonene, undecanal, and geraniol vapors, and differentiate between their smells utilizing the PL response pattern. This novel paper-based optical biosensor provides data in real-time, and is recoverable and suitable for working at room temperature and in a wide range of humidity levels. This platform opens new avenues for real-time sensing of volatile chemical compounds, odors, and flavors.

Species-independent analytical tools for next-generation agriculture.

Innovative approaches are urgently required to alleviate the growing pressure on agriculture to meet the rising demand for food. A key challenge for plant biology is to bridge the notable knowledge gap between our detailed understanding of model plants grown under laboratory conditions and the agriculturally important crops cultivated in fields or production facilities. This Perspective highlights the recent development of new analytical tools that are rapid and non-destructive and provide tissue-, cell- or organelle-specific information on living plants in real time, with the potential to extend across multiple species in field applications. We evaluate the utility of engineered plant nanosensors and portable Raman spectroscopy to detect biotic and abiotic stresses, monitor plant hormonal signalling as well as characterize the soil, phytobiome and crop health in a non- or minimally invasive manner. We propose leveraging these tools to bridge the aforementioned fundamental gap with new synthesis and integration of expertise from plant biology, engineering and data science. Lastly, we assess the economic potential and discuss implementation strategies that will ensure the acceptance and successful integration of these modern tools in future farming practices in traditional as well as urban agriculture.

Fabrication of Second Skin from Keratin and Melanin.

Second skin is a topically applied, skin-conforming material that mimics human skin properties and bears potential cosmetic and e-skin applications. To successfully integrate with natural skin, characteristics such as color and skin features must be matched. In this work, we prepared bio-based skin-like films from cross-linked keratin/melanin films (KMFs), using a simple fabrication method and non-toxic materials. The films retained their stability in aqueous solutions, showed skin-like mechanical properties, and were homogenous and handleable, with non-granular surfaces and a notable cross-linked structure as determined by attenuated total reflection (ATR). In addition, the combination of keratin and melanin allowed for adjustable tones similar to those of natural human skin. Furthermore, KMFs showed light transmittance and UV-blocking (up to 99%) as a function of melanin content. Finally, keratin/melanin ink (KMI) was used to inkjet-print high-resolution images with natural skin pigmented features. The KMFs and KMI may offer advanced solutions as e-skin or cosmetics platforms.