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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Halophyte biorefinery for polyhydroxyalkanoates production from Ulva sp. Hydrolysate with Haloferax mediterranei in pneumatically agitated bioreactors and ultrasound harvesting.

The present study tested the outdoor cultivation of Haloferax mediterranei for PHA production from green macroalgae Ulva sp. in pneumatically agitated bioreactors and applied ultrasonic separation for enhanced settling of archaeal cells. Scaled-up cultivation (40 L) yielded maximum biomass productivity of 50.1 ± 0.11 mg·L-1·h-1 with a PHA productivity of 27 ± 0.01 mg·L-1·h-1 and conversion yield of 0.107 g PHA per gram UlvaDW. The maximum mass fraction of PHA achieved in biomass was calculated to be 56% w/w. Ultrasonic harvesting of Hfx. mediterranei cells approached 30% removal at energy inputs around 7.8 kWh·m-3, and indicated no significant aggregation enhancement by Ca2+ addition. Molecular weight analysis showed an increase in Polydispersity Index (PDI) when the corresponding air velocities were increased suggesting that the polymer was more homogeneous at lower mixing velocities. The current study demonstrated scalable processes for PHA production using Ulva sp. feedstock providing new technologies for halophilic biorefinery.

Fighting SARS-CoV-2 with green seaweed Ulva sp. extract: extraction protocol predetermines crude ulvan extract anti-SARS-CoV-2 inhibition properties in in vitro Vero-E6 cells assay.

Due to the global COVID-19 pandemic, there is a need to screen for novel compounds with antiviral activity against SARS-COV-2. Here we compared chemical composition and the in vitro anti- SARS-COV-2 activity of two different Ulva sp. crude ulvan extracts: one obtained by an HCl-based and another one by ammonium oxalate-based (AOx) extraction protocols. The composition of the crude extracts was analyzed and their antiviral activity was assessed in a cytopathic effect reduction assay using Vero E6 cells. We show that the extraction protocols have a significant impact on the chemical composition, anti- SARS-COV-2 activity, and cytotoxicity of these ulvan extracts. The ulvan extract based on the AOx protocol had a higher average molecular weight, higher charge, and 11.3-fold higher antiviral activity than HCl-based extract. Our results strongly suggest that further bioassay-guided investigation into bioactivity of compounds found in Ulva sp. ulvan extracts could lead to the discovery of novel anti-SARS-CoV-2 antivirals.

Multi-scale modeling of intensive macroalgae cultivation and marine nitrogen sequestration.

Multi-scale macroalgae growth models are required for the efficient design of sustainable, economically viable, and environmentally safe farms. Here, we develop a multi-scale model for Ulva sp. macroalgae growth and nitrogen sequestration in an intensive cultivation farm, regulated by temperature, light, and nutrients. The model incorporates a range of scales by incorporating spatial effects in two steps: light extinction at the reactor scale (1 m) and nutrient absorption at the farm scale (1 km). The model was validated on real data from an experimental reactor installed in the sea. Biomass production rates, chemical compositions, and nitrogen removal were simulated under different seasons, levels of dilution in the environment and water-exchange rate in the reactor. This multi-scale model provides an important tool for environmental authorities and seaweed farmers who desire to upscale to large bioremediation and/or macroalgae biomass production farms, thus promoting the marine sustainable development and the macroalgae-based bioeconomy.

Seaweed cellulose scaffolds derived from green macroalgae for tissue engineering.

Extracellular matrix (ECM) provides structural support for cell growth, attachments and proliferation, which greatly impact cell fate. Marine macroalgae species Ulva sp. and Cladophora sp. were selected for their structural variations, porous and fibrous respectively, and evaluated as alternative ECM candidates. Decellularization-recellularization approach was used to fabricate seaweed cellulose-based scaffolds for in-vitro mammalian cell growth. Both scaffolds were confirmed nontoxic to fibroblasts, indicated by high viability for up to 40 days in culture. Each seaweed cellulose structure demonstrated distinct impact on cell behavior and proliferation rates. The Cladophora sp. scaffold promoted elongated cells spreading along its fibers' axis, and a gradual linear cell growth, while the Ulva sp. porous surface, facilitated rapid cell growth in all directions, reaching saturation at week 3. As such, seaweed-cellulose is an environmentally, biocompatible novel biomaterial, with structural variations that hold a great potential for diverse biomedical applications, while promoting aquaculture and ecological agenda.

Marine bacteria associated with the green seaweed Ulva sp. for the production of polyhydroxyalkanoates.

This work aimed to isolate a series of bacterial strains associated with the green seaweed Ulva sp. and evaluate their capability to manufacture PHA. The effect of the type of supplemented sugars found to be in macroalgae, on the growth and PHA productivity of the strains was studied. Analysis of the 16S rRNA gene sequence of the isolated strains revealed that the PHA-producing bacteria were phylogenetically related to the genus Cobetia, Bacillus, Pseudoaltermonas and Sulfitobacter, which showed high PHA contents among the isolates. The highest PHA content was observed in the case of Cobetia strain, with up to 61% w/w in the presence of mannitol and 12% w/w on Ulva sp. acid hydrolysate as a substrate.

Polyhydroxyalkanoates and biochar from green macroalgal Ulva sp. biomass subcritical hydrolysates: Process optimization and a priori economic and greenhouse emissions break-even analysis.

Although macroalgae biomass is an emerging sustainable feedstock for biorefineries, the optimum process parameters for their hydrolysis and fermentation are still not known. In the present study, the simultaneous production of polyhydroxyalkanoates (PHA) and biochar from green macroalgae Ulva sp. is examined, applying subcritical water hydrolysis and Haloferax mediterranei fermentation. First, the effects of temperature, treatment time, salinity, and solid load on the biomass and PHA productivity were optimized following the Taguchi method. Hydrolysis at 170 °C, 20 min residence time, 38 g L-1 salinity with a seaweed solid load of 5% led to the maximum PHA yield of 0.104 g g-1Ulva and a biochar yield of 0.194 ± 1.23 g g-1Ulva. Second, the effect of different initial culture densities on the biomass and PHA productivity was studied. An initial culture density of 50 g L-1 led to the maximum volumetric PHA productivity of 0.024 ± 0.002 g L-1 h-1 with a maximum PHA content of 49.38 ± 0.3% w/w Sensitivity analysis shows that within 90% confidence, the annual PHA production from Ulva sp. is 148.14 g PHA m-2 year-1 with an annual biochar production of 42.6 g m-2 year-1. Priori economic and greenhouse gas break-even analyses of the process were done to estimate annual revenues and allowable greenhouse gas emissions. The study illustrates that PHA production from seaweed hydrolysate using extreme halophiles coupled to biochar production could become a benign and promising step in a marine biorefinery.

High-Voltage, Pulsed Electric Fields Eliminate Pseudomonas aeruginosa Stable Infection in a Mouse Burn Model.

Objective: The incidence of severe infectious complications after burn injury increases mortality by 40%. However, traditional approaches for managing burn infections are not always effective. High-voltage, pulsed electric field (PEF) treatment shortly after a burn injury has demonstrated an antimicrobial effect in vivo; however, the working parameters and long-term effects of PEF treatment have not yet been investigated. Approach: Nine sets of PEF parameters were investigated to optimize the applied voltage, pulse duration, and frequency or pulse repetition for disinfection of Pseudomonas aeruginosa infection in a stable mouse burn wound model. The bacterial load after PEF administration was monitored for 3 days through bioluminescence imaging. Histological assessments and inflammation response analyses were performed at 1 and 24 h after the therapy. Results: Among all tested PEF parameters, the best disinfection efficacy of P. aeruginosa infection was achieved with a combination of 500 V, 100 µs, and 200 pulses delivered at 3 Hz through two plate electrodes positioned 1 mm apart for up to 3 days after the injury. Histological examinations revealed fewer inflammatory signs in PEF-treated wounds compared with untreated infected burns. Moreover, the expression levels of multiple inflammatory-related cytokines (interleukin [IL]-1α/ß, IL-6, IL-10, leukemia inhibitory factor [LIF], and tumor necrosis factor-alpha [TNF-α]), chemokines (macrophage inflammatory protein [MIP]-1α/ß and monocyte chemoattractant protein-1 [MCP-1]), and inflammation-related factors (vascular endothelial growth factor [VEGF], macrophage colony-stimulating factor [M-CSF], and granulocyte-macrophage colony-stimulating factor [G-CSF]) were significantly decreased in the infected burn wound after PEF treatment. Innovation: We showed that PEF treatment on infected wounds reduces the P. aeruginosa load and modulates inflammatory responses. Conclusion: The data presented in this study suggest that PEF treatment is a potent candidate for antimicrobial therapy for P. aeruginosa burn infections.