The green seaweed Ulva: tomorrow's "wheat of the sea" in foods, feeds, nutrition, and biomaterials.

Ulva, a genus of green macroalgae commonly known as sea lettuce, has long been recognized for its nutritional benefits for food and feed. As the demand for sustainable food and feed sources continues to grow, so does the interest in alternative, plant-based protein sources. With its abundance along coastal waters and high protein content, Ulva spp. have emerged as promising candidates. While the use of Ulva in food and feed has its challenges, the utilization of Ulva in other industries, including in biomaterials, biostimulants, and biorefineries, has been growing. This review aims to provide a comprehensive overview of the current status, challenges and opportunities associated with using Ulva in food, feed, and beyond. Drawing on the expertise of leading researchers and industry professionals, it explores the latest knowledge on Ulva's nutritional value, processing methods, and potential benefits for human nutrition, aquaculture feeds, terrestrial feeds, biomaterials, biostimulants and biorefineries. In addition, it examines the economic feasibility of incorporating Ulva into aquafeed. Through its comprehensive and insightful analysis, including a critical review of the challenges and future research needs, this review will be a valuable resource for anyone interested in sustainable aquaculture and Ulva's role in food, feed, biomaterials, biostimulants and beyond.

High-throughput lipidomic profiles sampled with electroporation-based biopsy differentiate healthy skin, cutaneous squamous cell carcinoma, and basal cell carcinoma.

BACKGROUND: The incidence rates of cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC) skin cancers are rising, while the current diagnostic process is time-consuming. We describe the development of a novel approach to high-throughput sampling of tissue lipids using electroporation-based biopsy, termed e-biopsy. We report on the ability of the e-biopsy technique to harvest large amounts of lipids from human skin samples. MATERIALS AND METHODS: Here, 168 lipids were reliably identified from 12 patients providing a total of 13 samples. The extracted lipids were profiled with ultra-performance liquid chromatography and tandem mass spectrometry (UPLC-MS-MS) providing cSCC, BCC, and healthy skin lipidomic profiles. RESULTS: Comparative analysis identified 27 differentially expressed lipids (p < 0.05). The general profile trend is low diglycerides in both cSCC and BCC, high phospholipids in BCC, and high lyso-phospholipids in cSCC compared to healthy skin tissue samples. CONCLUSION: The results contribute to the growing body of knowledge that can potentially lead to novel insights into these skin cancers and demonstrate the potential of the e-biopsy technique for the analysis of lipidomic profiles of human skin tissues.

Transmission of Genetic Material and Malignant Cells of Cutaneous Origin via Surgical Sutures.

BACKGROUND: Seeding of skin cancer cells following diagnostic or therapeutic surgical procedures can occur and might cause local recurrences. As current preferred therapy for skin malignancy is surgical excision, seeding of tumour cells by manipulating malignant tissue or suturing can be another factor leading to recurrences. OBJECTIVE: To evaluate whether genetic material and malignant cells adhere to standard suture materials. METHODS: This prospective study included patients who underwent excision of skin lesions. Monofilament and braided sutures were examined. Sutures were passed through the observed tumour or healthy skin margins and were examined for DNA material and cells by cytological analysis, cell culture and characterization, and DNA analysis. RESULTS: Twenty-two patients and 148 sutures were included. DNA quantification showed DNA material on all sutures, with no significant difference between braided and monofilament sutures. Cytological analysis showed that all slides prepared from cell blocks contained normal squamous and atypical cells. Cell culture and characterization showed viable cells adhering to the sutures under direct light microscopy. Cell cultures showed rapid proliferation of epithelial cells from squamous cell carcinoma specimens. CONCLUSION: Suture materials carry DNA material and cells, including malignant cells of cutaneous origin and may seed them at distant sites.

Allergic to bureaucracy? Regulatory allergenicity assessments of novel food: Motivations, challenges, compromises, and possibilities.

New sources of proteins are essential to meet the demands of the growing world population and evolving food trends. Assessing the allergenicity of proteins in novel food (NF) poses a significant food safety regulatory challenge. The Codex Alimentarius Commission presented an allergenicity assessment protocol for genetically modified (GM) foods, which can also be adapted for NF. Since no single laboratory test can adequately predict the allergenic potential of NF, the protocol follows a weight-of-evidence approach, evaluated by experts, as part of a risk management process. Regulatory bodies worldwide have adopted this safety protocol, which, among other things, promotes global harmonization. This review unravels the reliability and various motivations, terms, concepts, and approaches of allergenicity assessments, aiming to enhance understanding among manufacturers and the public. Health Canada, Food Safety Commission JAPAN, and Food Standards Australia New Zealand were surveyed, focusing on the European Food Safety Authority and the US Food Safety Administration for examples of scientific opinions regarding allergenicity assessments for novel and GM foods, from 2019 to 2023. According to our findings, current regulatory allergenicity assessments for NF approval primarily rely on literature reviews. Only a few of the NF assessments proactively presented additional tests. We recommend conducting bioinformatic analyses on NF when a panel of experts deems that there is insufficient prior scientific research.

Exergy efficiency of light conversion into biomass in the macroalga Ulva sp. (Chlorophyta) cultivated under the pulsed light in a photobioreactor.

Marine macroalgae are a potential feedstock for biorefineries that can reduce dependence on fossil fuels and contribute to bioeconomy. New knowledge and technologies for efficient conversion of solar energy into macroalgae biomass are needed to increase biomass yields and energy conversion efficiency. In this work, we show that the green macroalgae from Ulva sp. can grow under the pulsed light in a photobioreactor with higher exergy conversion efficiency in comparison to cultivation under constant light with the same intensity. In the tested frequencies, 1-40 Hz and duty cycles (DC) 1-100%, DC has a stronger impact on the growth rate than frequency. The efficiency of light transformation into biomass increased with decreasing DC. Pulsating with DC 20% led to 60% of the biomass chemical energy yield for the respective constant light (DC 100%). Models of Ulva sp. growth rate and exergy conversion efficiency as a function of pulsating light parameters were developed. These results open new directions to enhance solar to chemical energy conversion through macroalgae by controlling the light distribution in the macroalgal biomass.

Single-step electrical field strength screening to determine electroporation induced transmembrane transport parameters.

The design of effective electroporation protocols for molecular delivery applications requires the determination of transport parameters including diffusion coefficient, membrane resealing, and critical electric field strength for electroporation. The use of existing technologies to determine these parameters is time-consuming and labor-intensive, and often results in large inconsistencies in parameter estimation due to variations in the protocols and setups. In this work, we suggest using a set of concentric electrodes to screen a full range of electric field strengths in a single test to determine the electroporation-induced transmembrane transport parameters. Using Calcein as a fluorescent probe, we developed analytical methodology to determine the transport parameters based on the electroporation-induced pattern of fluorescence loss from cells. A monolayer of normal human dermal fibroblast (NHDF) cells were pre-loaded with Calcein and electroporated with an applied voltage of 750V with 10 and 50 square pulses with 50µs duration. Using our analytical model, the critical electric field strength for electroporation was found for the 10 and 50 pulses experiments. An inverse correlation between the field strength and the molecular transport time decay constant, and a direct correlation between field strength and the membrane permeability were observed. The results of this work can simplify the development of electroporation-assisted technologies for research and therapies.

Eradication of multidrug-resistant pseudomonas biofilm with pulsed electric fields.

Biofilm formation is a significant problem, accounting for over eighty percent of microbial infections in the body. Biofilm eradication is problematic due to increased resistance to antibiotics and antimicrobials as compared to planktonic cells. The purpose of this study was to investigate the effect of Pulsed Electric Fields (PEF) on biofilm-infected mesh. Prolene mesh was infected with bioluminescent Pseudomonas aeruginosa and treated with PEF using a concentric electrode system to derive, in a single experiment, the critical electric field strength needed to kill bacteria. The effect of the electric field strength and the number of pulses (with a fixed pulse length duration and frequency) on bacterial eradication was investigated. For all experiments, biofilm formation and disruption were confirmed with bioluminescent imaging and Scanning Electron Microscopy (SEM). Computation and statistical methods were used to analyze treatment efficiency and to compare it to existing theoretical models. In all experiments 1500 V are applied through a central electrode, with pulse duration of 50 µs, and pulse delivery frequency of 2 Hz. We found that the critical electric field strength (Ecr) needed to eradicate 100-80% of bacteria in the treated area was 121 ± 14 V/mm when 300 pulses were applied, and 235 ± 6.1 V/mm when 150 pulses were applied. The area at which 100-80% of bacteria were eradicated was 50.5 ± 9.9 mm(2) for 300 pulses, and 13.4 ± 0.65 mm(2) for 150 pulses. 80% threshold eradication was not achieved with 100 pulses. The results indicate that increased efficacy of treatment is due to increased number of pulses delivered. In addition, we that showed the bacterial death rate as a function of the electrical field follows the statistical Weibull model for 150 and 300 pulses. We hypothesize that in the clinical setting, combining systemic antibacterial therapy with PEF will yield a synergistic effect leading to improved eradication of mesh infections.

Electroporation-based technologies for medicine: principles, applications, and challenges.

When high-amplitude, short-duration pulsed electric fields are applied to cells and tissues, the permeability of the cell membranes and tissue is increased. This increase in permeability is currently explained by the temporary appearance of aqueous pores within the cell membrane, a phenomenon termed electroporation. During the past four decades, advances in fundamental and experimental electroporation research have allowed for the translation of electroporation-based technologies to the clinic. In this review, we describe the theory and current applications of electroporation in medicine and then discuss current challenges in electroporation research and barriers to a more extensive spread of these clinical applications.

An automated image processing method to quantify collagen fibre organization within cutaneous scar tissue.

Standard approaches to evaluate scar formation within histological sections rely on qualitative evaluations and scoring, which limits our understanding of the remodelling process. We have recently developed an image analysis technique for the rapid quantification of fibre alignment at each pixel location. The goal of this study was to evaluate its application for quantitatively mapping scar formation in histological sections of cutaneous burns. To this end, we utilized directional statistics to define maps of fibre density and directional variance from Masson's trichrome-stained sections for quantifying changes in collagen organization during scar remodelling. Significant increases in collagen fibre density are detectable soon after burn injury in a rat model. Decreased fibre directional variance in the scar was also detectable between 3 weeks and 6 months after injury, indicating increasing fibre alignment. This automated analysis of fibre organization can provide objective surrogate endpoints for evaluating cutaneous wound repair and regeneration.

Electroporation-Based Biopsy Treatment Planning with Numerical Models and Tissue Phantoms.

Molecular sampling with vacuum-assisted tissue electroporation is a novel, minimally invasive method for molecular profiling of solid lesions. In this paper, we report on the design of the battery-powered pulsed electric field generator and electrode configuration for an electroporation-based molecular sampling device for skin cancer diagnostics. Using numerical models of skin electroporation corroborated by the potato tissue phantom model, we show that the electroporated tissue volume, which is the maximum volume for biomarker sampling, strongly depends on the electrode's geometry, needle electrode skin penetration depths, and the applied pulsed electric field protocol. In addition, using excised human basal cell carcinoma (BCC) tissues, we show that diffusion of proteins out of human BCC tissues into water strongly depends on the strength of the applied electric field and on the time after the field application. The developed numerical simulations, confirmed by experiments in potato tissue phantoms and excised human cancer lesions, provide essential tools for the development of electroporation-based molecular markers sampling devices for personalized skin cancer diagnostics.

Regeneration and control of human fibroblast cell density by intermittently delivered pulsed electric fields.

Proliferative scarring is a human disease with neither available effective treatment nor relevant animal model. One of the hypotheses for scar formation involves deregulation of fibroblast signaling and delayed apoptosis. Here, we introduce a new chemical-free method for fibroblast density control in culture by intermittently delivered pulsed electric fields (IDPEF), which cause irreversible damage to cell membranes. Using 5-100 pulses with electric field strength of 150 V/mm, pulse duration 70 µs, and frequency of 1 Hz, we investigated the effects of PEF application on growth, death, and regeneration of normal human dermal fibroblasts in culture. We found that the fraction of fibroblasts that survive depends on the number of pulses applied and follows a Weibull distribution. We have successfully developed an IDPEF protocol that controls fibroblasts density in culture. Specifically, through application of IDPEF every 72 h for 12 days, we maintain a normal human dermal fibroblast density in the 3.1 ± 0.2 × 10(5) -1.4 ± 0.2 × 10(5) cell/mL range. Our results suggest that IDPEFs may prove useful as a non-chemical method for fibroblast density control in human wound healing.

Effects of season, depth and pre-cultivation fertilizing on Ulva growth dynamics offshore the Eastern Mediterranean Sea.

Offshore macroalgae production could provide an alternative source of biomass for food, materials and energy. However, the offshore environment in general, specifically the Eastern Mediterranean Sea (EMS) offshore, is a high energy and low nutrients environment, thus challenging for macroalgae farming. In this study, we experimentally investigated the impact of season, depth, and pre-cultivation fertilization duration on the growth rates and chemical composition of offshore Ulva biomass, and developed a predictive model tailored to offshore conditions, capable of estimating both biomass growth rate and nitrogen content. Specifically, we measured Ulva biomass growth rate and internal nitrogen in the nitrogen-poor EMS a few kilometers offshore the Israeli coast at various depths and on-shore pre-cultivation fertilization schedules. Based on these data, we constructed a predictive cultivation model of Ulva offshore growth, which allows for the optimization of fertilization requirements for offshore cultivation. This study provides new insights on the effects of seasonality, depth, and pre-cultivation fertilization duration on growth rates and chemical composition of offshore Ulva sp. biomass production.

Avocado seed waste bioconversion into poly(3-hydroxybutyrate) by using Cobetia amphilecti and ethyl levulinate as a green extractant.

The avocado processing industry produces up to 1.3M tons of agro-waste annually. Chemical analysis of avocado seed waste (ASW) revealed that it is rich in carbohydrates (464.7 ± 21.4 g kg-1) and proteins (37.2 ± 1.5 g kg-1). Optimized microbial cultivation of Cobetia amphilecti using an acid hydrolysate of ASW, generated poly(3-hydroxybutyrate) (PHB) in a 2.1 ± 0.1 g L-1 concentration. The PHB productivity of C. amphilecti cultivated on ASW extract was 17.5 mg L-1 h-1. The process in which a novel ASW substrate was utilized has been further augmented by using ethyl levulinate as a sustainable extractant. This process achieved 97.4 ± 1.9 % recovery yield and 100 ± 1 % purity (measured by TGA, NMR, and FTIR) of the target PHB biopolymer, along with a high and relatively uniform PHB molecular weight (Mw = 1831 kDa, Mn = 1481 kDa, Mw/Mn = 1.24) (measured by gel permeation chromatography), compared to PHB polymer extracted by chloroform (Mw = 389 kDa, Mn = 297 kDa, Mw/Mn = 1.31). This is the first example of ASW utilization as a sustainable and inexpensive substrate for PHB biosynthesis and ethyl levulinate as an efficient and green extractant of PHB from a single bacterial biomass.

Waste animal fat with hydrothermal liquefaction as a potential route to marine biofuels.

Unused animal waste rendered fat is a potential feedstock for marine biofuels. In this work, bio-oil was generated using hydrothermal liquefaction (HTL) of nitrogen-free and low sulfur rendered bovine fat. Maximum bio-oil yield of 28 ± 1.5% and high heating value of 38.5 ± 0.16 MJ·kg‒1 was obtained at 330 °C at 50% animal fat solid load and 20 min retention time. The nitrogen and sulfur content were negligible, making the produced bio-oil useful marine biofuel, taking into account current stringent regulations on NOx and SOx emissions. The economic analysis of the process, where part of the bovine fat waste is converted to the bio-oil and the semi-solid residues can be used to supply the heat demand of the HTL process and alternately generate electricity, showed that our process is likely to generate a positive profit margin on a large scale. We also showed the growing economic importance of electricity in the revenues as commercial production becomes more energy efficient.

Exploring multisite heterogeneity of human basal cell carcinoma proteome and transcriptome.

Basal cell carcinoma (BCC) is the most common type of skin cancer. Due to multiple, potential underlying molecular tumor aberrations, clinical treatment protocols are not well-defined. This study presents multisite molecular heterogeneity profiles of human BCC based on RNA and proteome profiling. Three areas from lesions excised from 9 patients were analyzed. The focus was gene expression profiles based on proteome and RNA measurements of intra-tumor heterogeneity from the same patient and inter-tumor heterogeneity in nodular, infiltrative, and superficial BCC tumor subtypes from different patients. We observed significant overlap in intra- and inter-tumor variability of proteome and RNA expression profiles, showing significant multisite heterogeneity of protein expression in the BCC tumors. Inter-subtype analysis has also identified unique proteins for each BCC subtype. This profiling leads to a deeper understanding of BCC molecular heterogeneity and potentially contributes to developing new sampling tools for personalized diagnostics therapeutic approaches to BCC.

Extract from the Macroalgae Ulva rigida Induces Table Grapes Resistance to Botrytis cinerea.

Fungal pathogens are a central cause of the high wastage rates of harvested fruit and vegetables. Seaweeds from the genus Ulva are fast-growing edible green macroalgae whose species can be found on the shore of every continent, and therefore present a resource that can be utilized on a global scale. In this study, we found that the application of ulvan extract, a sulfated polysaccharide extracted from Ulva rigida (1000 mg/L), elicited table grapes defense and reduced the incidence and decay area of Botrytis cinerea by 43% and 41%, respectively. In addition, compared to the control group at two days post-treatment, ulvan extract elicited a variety of defense-related biomarkers such as a 43% increase in the activity of reactive oxygen species, 4-fold increase in the activity of catalase, 2-fold increase in the activity of superoxide dismutase and 1.4-fold increase in the activity of chitinase. No increase was observed in phenylalanine ammonia-lyase activity, and the treatment did not affect fruit quality parameters such as the pH levels, sugar levels, and titratable acidity of grapes. These results illustrate the potential of ulvan extract to naturally induce the plant defense response and to reduce postharvest decay.

Turning mannitol-rich agricultural waste to poly(3-hydroxybutyrate) with Cobetia amphilecti fermentation and recovery with methyl levulinate as a green solvent.

The present study explored the use of mannitol and mannitol-rich agro-industrial wastes as substrates for PHB production by Cobetia amphilecti isolated from the green Ulva sp. seaweed. Cultivation of C. amphilecti on mannitol, celery, and olive leaves (OLs) waste led to 4.20, 6.00, and 5.16 g L-1 of cell dry mass (CDM), 76.3, 25.5, and 12.0% of PHB content in CDM and 3.2, 1.53, and 0.62 g L-1 of PHB concentration, respectively; which suggested that they can be exploited as carbon substrates for the production of PHB. Extraction of PHB from C. amphilecti cultures by solubilization in the green solvent methyl levulinate (ML) (2% w/w, 140 °C, 1 h) indicated that the recovery yield and purity of PHB are above 97 and 90% w/w, respectively. The use of ML could be an attractive method for the recovery of PHB when safe and non-toxic solvents are required.

Nondestructive protein sampling with electroporation facilitates profiling of spatial differential protein expression in breast tumors in vivo.

Excision tissue biopsy, while central to cancer treatment and precision medicine, presents risks to the patient and does not provide a sufficiently broad and faithful representation of the heterogeneity of solid tumors. Here we introduce e-biopsy-a novel concept for molecular profiling of solid tumors using molecular sampling with electroporation. As e-biopsy provides access to the molecular composition of a solid tumor by permeabilization of the cell membrane, it facilitates tumor diagnostics without tissue resection. Furthermore, thanks to its non tissue destructive characteristics, e-biopsy enables probing the solid tumor multiple times in several distinct locations in the same procedure, thereby enabling the spatial profiling of tumor molecular heterogeneity.We demonstrate e-biopsy in vivo, using the 4T1 breast cancer model in mice to assess its performance, as well as the inferred spatial differential protein expression. In particular, we show that proteomic profiles obtained via e-biopsy in vivo distinguish the tumors from healthy breast tissue and reflect spatial tumor differential protein expression. E-biopsy provides a completely new molecular sampling modality for solid tumors molecular cartography, providing information that potentially enables more rapid and sensitive detection at lesser risk, as well as more precise personalized medicine.

Electroporation-based proteome sampling ex vivo enables the detection of brain melanoma protein signatures in a location proximate to visible tumor margins.

A major concern in tissue biopsies with a needle is missing the most lethal clone of a tumor, leading to a false negative result. This concern is well justified, since needle-based biopsies gather tissue information limited to needle size. In this work, we show that molecular harvesting with electroporation, e-biopsy, could increase the sampled tissue volume in comparison to tissue sampling by a needle alone. Suggested by numerical models of electric fields distribution, the increased sampled volume is achieved by electroporation-driven permeabilization of cellular membranes in the tissue around the sampling needle. We show that proteomic profiles, sampled by e-biopsy from the brain tissue, ex vivo, at 0.5mm distance outside the visible margins of mice brain melanoma metastasis, have protein patterns similar to melanoma tumor center and different from the healthy brain tissue. In addition, we show that e-biopsy probed proteome signature differentiates between melanoma tumor center and healthy brain in mice. This study suggests that e-biopsy could provide a novel tool for a minimally invasive sampling of molecules in tissue in larger volumes than achieved with traditional needle biopsies.

Pulsed Electric Fields Induce Extracellular Matrix Remodeling through Matrix Metalloproteinases Activation and Decreased Collagen Production.

Impairment of extracellular matrix remodeling is observed in the tumor microenvironment or fibrosis and results in excessive collagen production and/or decreased degradation by matrix metalloproteinases (MMPs). Thanks to their local application and transient effects, physical stimuli appear as attractive tools to remodel the extracellular matrix. We assessed the potential of pulsed electric field technology, classically applied to drug delivery, to induce collagen remodeling at the tissue scale. A sophisticated in vitro tissue-engineered human dermal substitute was used to show that microsecond and millisecond pulsed electric fields induced (i) a rapid modulation (4 hours after electrostimulation) of mRNA genes composing the matrisome, particularly a downregulation of procollagens and extracellular matrix maturation enzymes such as transglutaminase 2 and lysyl oxidase like; (ii) a transient decrease in procollagens production and hydroxyproline tissue content within a week after electrostimulation; (iii) a long-lasting ROS-dependent overactivation of matrix metalloproteinases for at least 48 hours; and (iv) a downregulation of TGFß1. These observations underpin that pulsed electric fields, a technology already approved for clinical use combined with anticancer agents, are particularly promising to provide local and effective treatment of abnormal extracellular matrix.