Reduction In Metabolic Activity Research Articles

Identification of early events in nitrogen mustard pulmonary toxicity that are independent of infiltrating inflammatory cells using precision cut lung slices

Nitrogen mustard (NM; mechlorethamine) is a cytotoxic vesicant known to cause acute lung injury which can progress to chronic disease. Due to the complex nature of NM injury, it has been difficult to analyze early responses of resident lung cells that initiate inflammation and disease progression. To investigate this, we developed a model of acute NM toxicity using murine precision cut lung slices (PCLS), which contain all resident lung cell populations. PCLS were exposed to NM (1–100 μM) for 0.5–3 h and analyzed 1 and 3 d later. NM caused a dose-dependent increase in cytotoxicity and a reduction in metabolic activity, as measured by LDH release and WST-1 activity, respectively. Optimal responses were observed with 50 μM NM after 1 h incubation and these conditions were used in further experiments. Analysis of PCLS bioenergetics using an Agilent Seahorse showed that NM impaired both glycolytic activity and mitochondrial respiration. This was associated with injury to the bronchial epithelium and a reduction in methacholine-induced airway contraction. NM was also found to cause DNA damage in bronchial epithelial cells in PCLS, as measured by expression of γ-H2AX, and to induce oxidative stress, which was evident by a reduction in glutathione levels and upregulation of the antioxidant enzyme catalase. Cleaved caspase-3 was also upregulated in airway smooth muscle cells indicating apoptotic cell death. Characterizing early events in NM toxicity is key in identifying therapeutic targets for the development of efficacious countermeasures.

Antimicrobial Activity of Methylene Blue Associated with Photodynamic Therapy: In Vitro Study in Multi-Species Oral Biofilm.

The control of infectious diseases caused by biofilms is a continuing challenge for researchers due to the complexity of their microbial structures and therapeutic implications. Photodynamic therapy as an adjunctive anti-infective treatment has been described as a possible valid approach but has not been tested in polymicrobial biofilm models. This study evaluated the effect of photodynamic therapy in vitro with methylene blue (MB) 0.01% and red LEDs (λ = 660 nm, power density ≈ 330 mW/cm2, 2 mm distance from culture) on the metabolic activity and composition of a multispecies subgingival biofilm. Test Groups LED and MB + LED showed a more significant reduction in metabolic activity than the non-LED application group (~50 and 55%, respectively). Groups LED and MB equally affected (more than 80%) the total bacterial count in biofilms. No differences were noted in the bacterial biofilm composition between the groups. In vitro LED alone or the MB + LED combination reduced the metabolic activity of bacteria in polymicrobial biofilms and the total subgingival biofilm count.

Labile assembly of a tardigrade protein induces biostasis.

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of fibrillization and gelation for CAHS D similar to that of intermediate filament assembly. We show that in vitro, gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fibrillar networks during osmotic stress. Fibrillar networking of CAHS D improves survival of osmotically shocked cells. We observe two emergent properties associated with fibrillization; (i) prevention of cell volume change and (ii) reduction of metabolic activity during osmotic shock. We find that there is no significant correlation between maintenance of cell volume and survival, while there is a significant correlation between reduced metabolism and survival. Importantly, CAHS D's fibrillar network formation is reversible and metabolic rates return to control levels after CAHS fibers are resolved. This work provides insights into how tardigrades induce reversible biostasis through the self-assembly of labile CAHS gels.

Lucilia sericata Maggot Extract: A Promising Tool against Biofilms of Antimicrobial Resistant Strains of Staphylococcus aureus and Pseudomonas aeruginosa

Background: This study explores the impact of Lucilia sericata maggots on the development and eradication of biofilms created by the pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa.
 Methods: We assessed the influence of Lucilia sericata maggot extract on the viability of planktonic bacteria, the formation and disruption of biofilms, bacterial metabolic activity. Also the effect of simultaneous ES-antibiotic treatment in biofilm elimination was investigated. Additionally, the expression levels of genes associated with biofilm formation, namely LasI, psLA, agrA, and icaD was studied.
 Results: The results showed that ES can reduce the viability of planktonic S. aureus, significantly. Furthermore, ES of larvae fed on S. aureus-infected meat displayed the most substantial inhibition of biofilm formation (62.11% and 75.04% inhibition for S. aureus and P. aeruginosa, respectively). A similar trend was observed in biofilm destruction, with values of 56.67% and 68.50% inhibition for S. aureus and P. aeruginosa, respectively. The simultaneous application of ES of larvae that fed on S. aureus-infected meat and the minimum inhibitory concentration (MIC) of gentamicin resulted in 100% inhibition of biofilm formation by S. aureus. Notably, the group treated with ES of larvae fed on S. aureus-infected meat exhibited the most significant reduction in metabolic activity, with values of 95.03% and 68.25% for S. aureus and P. aeruginosa, respectively. The expression of LAsI and pslA genes in P. aeruginosa and the expression of agrA and icaD genes in S. aureus has decreased
 Conclusion: The findings of this study demonstrate that maggot extract has not only impacted the formation, but also eliminated the biofilms of S. aureus and P. aeruginosa.

Impact of Nintedanib and Anti-Angiogenic Agents on Uveal Melanoma Cell Behavior.

The purpose of this study was to investigate the direct impact of the combined angiokinase inhibitor nintedanib as well as the anti-angiogenic agents ranibizumab, bevacizumab, and aflibercept on the primary uveal melanoma (UM) cell line Mel270 and liver metastasis UM cell line OMM2.5. The metabolic activity, viability, and oxidative stress levels were analyzed by the Thiazolyl Blue Tetrazolium Bromide (MTT), LIVE/DEAD, and reactive oxygen species (ROS) assays. Expression of intracellular VEGF-A165 and VEGF receptor-2 was detected by immunofluorescent staining. The secretion of VEGF-A165 into the cell culture supernatants was evaluated by VEGF-A165 ELISA. Nintedanib, at a concentration of 1µg/mL, resulted in a median reduction of metabolic activity (for Mel270 of approximately 38% and for OMM2.5 of 46% compared to the untreated control) without exerting toxicity in either cell line, whereas the other 3 substances did not result in any changes (which also means that none of the 4 substances led to an increased cell death). Moreover, nintedanib (1µg/mL) induced oxidative stress in the Mel270 by approximately 1.2 to 1.5-fold compared to the untreated control, but not the OMM2.5 cells. Nintedanib could suppress the growth of UM cells in a concentration-dependent manner. The metastatic UM cell line OMM2.5 was not sensitive to the pro-oxidant activity of nintedanib. This study was the first to investigate nintedanib in the context of UM. We propose further investigation of this substance to elucidate its effects on this tumor entity with the hope of identifying advantageous therapeutic options for future adjuvant tumor therapies.

Denture Adhesives Associated with Silver Vanadate: Antimicrobial Approach Against Multi- Species Biofilms on Acrylic Resin Surfaces.

Alternatives have been sought to add an antimicrobial property to denture adhesives. This study evaluated the antimicrobial potential of adhesives associated with nanostructured silver vanadate decorated with silver nanoparticles (β-AgVO3). Specimens in acrylic resin were treated with the adhesives associated with β-AgVO3 (1%, 2.5%, 5% and 10%). As control, specimens treated only with Ultra Corega Cream (UCC) or Ultra Corega Powder (UCP) adhesive were used. Multispecies biofilm of Candida albicans, Candida glabrata, Streptococcus mutans and Staphylococcus aureus was evaluated by counting colony forming units per milliliter (CFU/mL), colorimetric assay and fluorescence microscopy. The data were analyzed using the two-way analysis of variance (ANOVA) and Bonferroni multiple comparisons test (α=0.05). For both adhesives, a small amount of β-AgVO3 (1%) completely inhibited S. mutans (P⟨0.05). For the other microorganisms, there was a reduction in metabolic activity and complete inhibition in the groups with intermediate or greater amounts of nanomaterial (P⟨0.05), except for C. albicans, which was reduced (P⟨0.05) but not completely inhibited in UCP. Microscopy that showed less biofilm in the groups with β-AgVO3 and in the UCC than UCP. Denture adhesives in powder and cream form with β-AgVO3 showed potential antimicrobial activity against multispecies biofilm. Powder adhesive showed higher biofilm formation.

Vanadium(V) complexes derived from triphenylphosphonium and hydrazides: cytotoxicity evaluation and interaction with biomolecules.

The development of coordination compounds with antineoplastic therapeutic properties is currently focused on non-covalent interactions with deoxyribonucleic acid (DNA). Additionally, the interaction profiles of these compounds with globular plasma proteins, particularly serum albumin, warrant thorough evaluation. In this study, we report on the interactions between biomolecules and complexes featuring hydrazone-type imine ligands coordinated with vanadium. The potential to enhance the therapeutic efficiency of these compounds through mitochondrial targeting is explored. This targeting is facilitated by the derivatization of ligands with triphenylphosphonium groups. Thus, this work presents the synthesis, characterization, interactions, and cytotoxicity of dioxidovanadium(V) complexes (C1-C5) with a triphenylphosphonium moiety. These VV-species are coordinated to hydrazone-type iminic ligands derived from (3-formyl-4-hydroxybenzyl)triphenylphosphonium chloride ([AH]Cl) and aromatic hydrazides ([H2L1]Cl-[H2L5]Cl). The structures of the five complexes were elucidated through single-crystal X-ray diffraction and vibrational spectroscopies, confirming the presence of dioxidovanadium(V) species in various geometries with degrees of distortion (τ = 0.03-0.50) and highlighting their zwitterionic characteristics. The molecular structural stability of C1-C5 in solution was ascertained using 1H, 19F, 31P, and 51V-nuclear magnetic resonance. Moreover, their interactions with biomolecules were evaluated using diverse spectroscopic methodologies and molecular docking, indicating moderate interactions (Kb ≈ 104 M-1) with calf thymus DNA in the minor groove and with human serum albumin, predominantly in the superficial IB subdomain. Lastly, the cytotoxic potentials of these complexes were assessed in keratinocytes of the HaCaT lineage, revealing that C1-C5 induce a reduction in metabolic activity and cell viability through apoptotic pathways.

Comparative characteristics of neuropsychological and neurometabolic changes in patients with Alzheimer's disease and vascular cognitive impairment

To investigate the pattern and connections of neuropsychological and metabolic indices in patients with cognitive disorders of Alzheimer's and vascular (subcortical-cortical) types of different severity. A total of 177 patients were examined, including 85 patients with Alzheimer's disease (AD) and 92 patients with vascular cognitive impairment (VCI). All patients underwent complex neuropsychological examination; 18F-FDG PET was performed in 17 patients with AD and 15 patients with VCI. The greatest changes in patients with AD were noted in the mnestic sphere, and the indicators significantly differed from the results of the study of patients with VCI already at the pre-dementia stage. Neurodynamic and dysregulatory disorders prevailed in patients with VCI. Patients with AD showed bilateral symmetrical reduction of metabolic activity in the cortex of parietal and temporal lobes, often in combination with marked hypometabolism in the hippocampal region. In patients with VCI, there were areas of decreased brain tissue metabolism of different localization and size, mainly in the projection of the basal ganglia and in the prefrontal and parietal cortex, as well as in the cingulate gyrus, which indirectly confirms the mechanism of disconnection of subcortical and cortical structures. In AD, impaired metabolic activity in the hippocampal region correlated with impaired temporal and spatial orientation (ρ=-0.54, p<0.05), memory impairment (ρ=-0.71, p<0.005). Hypometabolism of the parietal lobe cortex was associated with total MMSE score (ρ=-0.8, p<0.001), 10-word test (ρ=-0.89, p<0.001 and ρ=-0.82, p<0.001), visual-spatial impairment (ρ=-0.64, p<0.01), categorical association test (ρ=-0.73, p<0.005). In patients with VCI, dysregulatory disorders correlated with hypometabolism in the thalamic projection (ρ=-0.56, p<0.05), prefrontal cortex (ρ=-0.64, p<0.05) and in the cingulate gyrus (anterior regions) (ρ=-0.53, p<0.05). The results indicate the presence of differences in cognitive impairment and cerebral metabolism in patients with AD and VCI.

Antibacterial evaluation of different prosthetic liner textiles coated by CuO nanoparticles

Prosthetic liners are mainly used as an interface between residual limbs and prosthetic sockets to minimize physical and biological damage to soft tissue. However, the closed and moist conditions within liners and the amputee's skin provide a suitable environment for bacterial growth to cause infections. This study aimed to coat a comprehensive variant material with copper oxide nanoparticles (CuO NPs) and compare their surface analysis and antibacterial properties. These materials were covered with CuO NPs solution at a concentration of 70 μg mL−1 to achieve this purpose. After drying, their surface characteristics were analyzed by measuring zeta potential, contact angle, surface roughness, and fiber arrangement. Cu-released concentration from the coatings into the acetate buffer solution by inductively coupled plasma mass spectrometry indicated that lycra and nylon quickly released Cu ions to concentrations up to ∼0.2 μg mL−1 after 24 h, causing low metabolic activity of human bone–marrow mesenchymal stem cells (bMSC) in the indirect assay. Antibacterial activity of the coated specimens was evaluated by infecting their surfaces with the Gram-positive bacteria Staphylococcus epidermidis, reporting a significant ∼40 % reduction of metabolic activity for x-dry after 24 h; in addition, the number of viable bacterial colonies adhered to the surface of this material was reduced by ∼23 times in comparison with non-treated x-dry that were visually confirmed by scanning electron microscope. In conclusion, CuO NPs x-dry shows optimistic results to pursue further experiments due to its slow speed of Cu release and prolonged antibacterial activity, as well as its compatibility with human cells.

Cu-doped bioactive glass with enhanced in vitro bioactivity and antibacterial properties

This work aimed to optimize, produce and characterize Cu-doped bioactive glasses which are antibacterial without the addition of antibiotics obtained via ion exchange in an aqueous solution. According to morphological, compositional and structural analyses, 0.001 M was selected as the most optimal concentration of the ion exchange solution. The doped glass was then compared to the undoped one to investigate the effect of Cu-doping on the glass surface composition and bioactivity. Cu-doping was found to enhance the bioactivity kinetics and the following hydroxyapatite formation, evidenced by X-ray diffraction, energy dispersive X-ray spectroscopy, and zeta potential measurements. Besides that, the zeta potential titration measurements confirmed that the Cu-doping did not alter the surface chemical stability of the glass both in the inflammatory and physiological pH range. Moreover, the leaching ability of Cu2+-ions, both in physiological and inflammatory-mimicking conditions, was measured, followed by an in-depth study of the antibacterial properties, using two protocols to distinguish between the antiadhesive, antibacterial, and antibiofilm effects. For both protocols, a reduction of metabolic activity and Colony-Forming Unit after 24 h against Staphylococcus aureus Multi-Drug resistance strain was evidenced. These results showed that Cu-doped glass could show potential as a bioactive and antibacterial surface for bone surgery applications.

The gut microbiome mediates adaptation to scarce food in Coleoptera.

Beetles are ubiquitous cave invertebrates worldwide that adapted to scarce subterranean resources when they colonized caves. Here, we investigated the potential role of gut microbiota in the adaptation of beetles to caves from different climatic regions of the Carpathians. The beetles' microbiota was host-specific, reflecting phylogenetic and nutritional adaptation. The microbial community structure further resolved conspecific beetles by caves suggesting microbiota-host coevolution and influences by local environmental factors. The detritivore species hosted a variety of bacteria known to decompose and ferment organic matter, suggesting turnover and host cooperative digestion of the sedimentary microbiota and allochthonous-derived nutrients. The cave Carabidae, with strong mandibula, adapted to predation and scavenging of animal and plant remains, had distinct microbiota dominated by symbiotic lineages Spiroplasma or Wolbachia. All beetles had relatively high levels of fermentative Carnobacterium and Vagococcus involved in lipid accumulation and a reduction of metabolic activity, and both features characterize adaptation to caves.

In Vitro Efficacy of Dalbavancin as a Long-Acting Anti-Biofilm Agent Loaded in Bone Cement.

Based on previous studies by our group in which we demonstrated that dalbavancin loaded in bone cement had good elution capacity for the treatment of biofilm-related periprosthetic infections, we now assess the anti-biofilm activity of dalbavancin and compare it with that of vancomycin over a 3-month period. We designed an in vitro model in which we calculated the percentage reduction in log cfu/mL counts of sonicated steel discs contaminated with staphylococci and further exposed to bone cement discs loaded with 2.5% or 5% vancomycin and dalbavancin at various timepoints (24 h, 48 h, 1 week, 2 weeks, 6 weeks, and 3 months). In addition, we tested the anti-biofilm activity of eluted vancomycin and dalbavancin at each timepoint based on a 96-well plate model in which we assessed the percentage reduction in metabolic activity. We observed a significant decrease in the dalbavancin concentration from 2 weeks of incubation, with sustained anti-biofilm activity up to 3 months. In the case of vancomycin, we observed a significant decrease at 1 week. The concentration gradually increased, leading to significantly lower anti-biofilm activity. The percentage reduction in cfu/mL counts was higher for dalbavancin than for vancomycin at both the 2.5% and the 5% concentrations. The reduction in log cfu/mL counts was higher for S. epidermidis than for S. aureus and was particularly more notable for 5% dalbavancin at 3 months. In addition, the percentage reduction in metabolic activity also decreased at 3 months in 5% dalbavancin and 5% vancomycin, with more notable values recorded for the latter.

Abstract P3043: Mir92b-3p: A Key Regulator In Vascular Homeostasis And Remodelling

In vascular disease, cells undergo important alterations in cellular function, influencing vascular homeostasis. Apoptosis is one of these functions, affecting vascular tone and plaque stability. Influencing this process for example, by targeting microRNAs is key for therapeutic strategies tackling vascular diseases in a cell-specific manner. We identified the critical role of miR92b-3p on the functional properties of vascular cells. Studying human primary coronary artery smooth muscle cells (SMCs) and human umbilical vein endothelial cells (ECs), methods included expression analysis in mice tissue after restenosis by wire-induced injury and in atherosclerosis. The role of miR-92b on cellular function and target regulation was determined by pre- and antagomir transfection. Initial analysis in vivo showed miR92b-3p to be significantly upregulated in atherosclerosis (p&lt;0,01) and regulated in restenosis (p&lt;0,05). Under growth conditions in both ECs and SMCs, miR-92b-3p was significantly higher expressed (p&lt;0,05). In SMCs, upregulation of miR92b-3p resulted in increased proliferation (p&lt;0,01) and significantly enhanced cell migration in SMCs (p&lt;0,001) but hampered migration in ECs (p&lt;0,01). Downregulation of miR-92b-3p in SMC resulted in decreased migrational (p&lt;0.001) and proliferative capacity (p&lt;0.001), accompanied by a reduction in metabolic activity (p&lt;0.05) and, exclusively in SMCs, increased apoptosis (p&lt;0.05). In ECs, proliferation was enhanced following downregulation (p&lt;0,01). Multiple targets were identified, using Western blot and qPCR including KLF4 (p&lt;0,01), DKK3 (p&lt;0,05), Notch1 (p&lt;0,01), PTEN (p&lt;0,05), and especially in SMC, Bim (p&lt;0,001). Regulation of Bim by miR92b-3p and subsequent apoptosis was then evaluated by siRNA-knockdown experiments, confirming their strong interaction. We further verified the crucial role of miR-92b-3p by reversing cell death in apoptotic SMCs through miR-92b-3p overexpression experiments (p&lt;0.05). This study reports miR 92b-3p to be a crucial regulator of vascular function. Therapeutic regulation might present a promising strategy for stabilizing critical plaques, restoring the function of vascular cells, and thereby preventing vascular remodeling.

Preparation and Characterization of Hydrogel Films and Nanoparticles Based on Low-Esterified Pectin for Anticancer Applications.

Prospective adjuvant anticancer therapy development includes the establishing of drug delivery systems based on biocompatible and biodegradable carriers. We have designed films and nanoparticles (NPs) based on low-esterified pectin hydrogel using the ionic gelation method. We investigated morphology, nanomechanical properties, biocompatibility and anticancer activity. Hydrogel films are characterized by tunable viscoelastic properties and surface nanoarchitectonics through pectin concentration and esterification degree (DE), expressed in variable pore frequency and diameter. An in vitro study showed a significant reduction in metabolic activity and the proliferation of the U87MG human glioblastoma cell line, probably affected via the adhesion mechanism. Glioma cells formed neurosphere-like conglomerates with a small number of neurites when cultured on fully de-esterified pectin films and they did not produce neurites on the films prepared on 50% esterified pectin. Pectin NPs were examined in terms of size distribution and nanomechanical properties. The NPs' shapes were proved spherical with a mean diameter varying in the range of 90-115 nm, and a negative zeta potential from -8.30 to -7.86 mV, which indicated their stability. The NPs did not demonstrate toxic effect on cells or metabolism inhibition, indicating good biocompatibility. Nanostructured biomaterials prepared on low-esterified pectins could be of interest for biomedical applications in adjuvant anticancer therapy and for designing drug delivery systems.

O-006 Novel and scalable non-invasive metabolic imaging of early embryos using a lab-on-a-chip approach

Abstract Study question Is it feasible to safely determine metabolic imaging signatures to measure nicotinamide adenine dinucleotide (NADH) associated auto-fluorescence in early embryos using a scalable lab-on-a-chip approach? Summary answer We developed an optofluidic device capable of non-invasively obtain high-resolution 3D images of the metabolic activity of live mouse embryos using a lab-on-a-chip approach. What is known already Selecting the most suitable embryos for implantation and subsequent healthy live birth is crucial to the success rate of assisted reproduction and offspring health. Thus, developing non-invasive methods that are reliable to assess oocyte and embryo quality has been a significant aim for assisted reproduction. Besides morphological evaluation using optical microscopy, a promising alternative is the non-invasive imaging of live embryos to establish metabolic activity performance. However, metabolic imaging has been only achieved using highly complex advanced microscopy such as Fluorescence-lifetime imaging (FLIM) and hyperspectral microscopy, methods that are costly and challenging, limiting the potential for deployment in fertility clinics. Study design, size, duration The non-invasive nature of the system was investigated by assessing the development and viability of live embryos after embryo culture for 67hrs post metabolic imaging at the 2-cell embryo stage (n = 115), including a control for culture conditions and sham controls (system no-illuminated). Embryo quality of developed blastocysts was assessed by immunocytochemistry to quantify trophectoderm and inner mass cells (n = 75). Furthermore, inhibition of metabolic activity (FK866 inhibitor) during embryo culture was also assessed (n = 18). Participants/materials, setting, methods Optofluidic devices were manufactured by cast-moulding using a negative photoresist (SU8-2075; MicroChemicals GmbH, Ulm, Germany) by a standard UV-photolithography process. The microstructures fabricated of polydimethylsiloxane (PDSM) integrated Light Sheet Fluorescence Microscopy into a microfluidic system, including on-chip micro-lenses to generate a light sheet at the center of a microchannel. Super-ovulated F1 (CBA/C57Bl6) mice were used to produce 2-cell embryos and embryo culture experiments. Blastocyst formation rates and embryo quality (immunocytochemistry) were compared between study groups. Main results and the role of chance The optofluidic device was capable of non-invasively obtaining high-resolution 3D images of the metabolic activity in live mouse embryos. The system’s design allowed continuous tracking of the embryo location, including high control displacement through the light-sheet, fast imaging of the embryos (&amp;lt;2 second) and keeping a low dose of light exposure (16 J cm-2 and 8 J cm-2). Optimum settings for keeping sample viability showed that a modest light dosage was capable to obtain 30 times higher signal-noise-ratio images than images obtained with a confocal system (p &amp;lt; 0.00001; t-test). The results showed no significant differences between the control, illuminated and non-illuminated embryos (sham control) for embryo development as well as embryo quality at the blastocyst stage (p &amp;gt; 0.05; Yate’s chi squared test). Additionally, embryos with inhibited metabolic activity showed decreased blastocyst formation rates as well as 47% reduction in metabolic activity measured by non-invasive metabolic imaging (p &amp;lt; 0.0001; t-test). This study reports an optofluidic device capable of non-invasive metabolic imaging of live embryos using a similar concept as previously reported using FLIM technology and hyperspectral microscopy, but allowing a novel, scalable and affordable system with implementation potential in standard IVF laboratory equipment. Limitations, reasons for caution The study was conducted using a mouse model focused on early embryo development. Further safety studies are required to assess embryonic health by investigating the impact of the use of light during embryo development, live birth, embryo gene expression and epigenome stability. Wider implications of the findings This lab-on-a-chip is novel, scalable and has the potential to be used in fertility clinics as a technology platform to enable real-time monitoring of the metabolic function of embryos prior embryo transfer. Future applications include potential integration into existing IVF laboratory equipment such as bench and time-lapse incubators. Trial registration number N/A

Cold Physical Plasma-Mediated Fenretinide Prodrug Activation Confers Additive Cytotoxicity in Epithelial Cells.

Cold physical plasma is a partially ionized gas operated at body temperature and utilized for heat-sensitive technical and medical purposes. Physical plasma is a multi-component system consisting of, e.g., reactive species, ions and electrons, electric fields, and UV light. Therefore, cold plasma technology is an interesting tool for introducing biomolecule oxidative modifications. This concept can be extended to anticancer drugs, including prodrugs, which could be activated in situ to enhance local anticancer effects. To this end, we performed a proof-of-concept study on the oxidative prodrug activation of a tailor-made boronic pinacol ester fenretinide treated with the atmospheric pressure argon plasma jet kINPen operated with either argon, argon-hydrogen, or argon-oxygen feed gas. Fenretinide release from the prodrug was triggered via Baeyer-Villiger-type oxidation of the boron-carbon bond based on hydrogen peroxide and peroxynitrite, which were generated by plasma processes and chemical addition using mass spectrometry. Fenretinide activation led to additive cytotoxic effects in three epithelial cell lines in vitro compared to the effects of cold plasma treatment alone regarding metabolic activity reduction and an increase in terminal cell death, suggesting that cold physical plasma-mediated prodrug activation is a new direction for combination cancer treatment studies.

Protease-Responsive Hydrogel Microparticles for Intradermal Drug Delivery.

Protease-responsive multi-arm polyethylene glycol-based microparticles with biscysteine peptide crosslinkers (CGPGG↓LAGGC) were obtained for intradermal drug delivery through inverse suspension photopolymerization. The average size of the spherical hydrated microparticles was ∼40 μm after crosslinking, making them attractive as a skin depot and suitable for intradermal injections, as they are readily dispensable through 27G needles. The effects of exposure to matrix metalloproteinase 9 (MMP-9) on the microparticles were evaluated by scanning electron microscopy and atomic force microscopy, demonstrating partial network destruction and decrease in elastic moduli. Given the recurring course of many skin diseases, the microparticles were exposed to MMP-9 in a flare-up mimicking fashion (multiple-time exposure), showing a significant increase in release of tofacitinib citrate (TC) from the MMP-responsive microparticles, which was not seen for the non-responsive microparticles (polyethylene glycol dithiol crosslinker). It was found that the degree of multi-arm complexity of the polyethylene glycol building blocks can be utilized to tune not only the release profile of TC but also the elastic moduli of the hydrogel microparticles, with Young's moduli ranging from 14 to 140 kPa going from 4-arm to 8-arm MMP-responsive microparticles. Finally, cytotoxicity studies conducted with skin fibroblasts showed no reduction in metabolic activity after 24 h exposure to the microparticles. Overall, these findings demonstrate that protease-responsive microparticles exhibit the properties of interest for intradermal drug delivery.

Examination of the potential of refrigerated seawater to improve live transport of the mussel Perna canaliculus: Physiological responses, meat quality and safety implications under different chilled storage conditions

Refrigerated seawater (RSW) based systems for post-harvest storage of green-lipped mussels (Perna canaliculus; also known as Greenshell™ mussels) were assessed. Two post-harvest storage trials were conducted in October 2020 and March 2021 (austral spring and summer respectively) on mussels recovered for 2.5 days after harvest in flow-through seawater. The mussels were randomly assigned to three post-harvest treatments, all conducted at 6.5 °C for 10 days: Chilled in air; immersion in recirculated RSW; and Periodic immersion in RSW (6 min h−1). A range of physiological, morphometric and quality measures were screened to assess their ability to indicate mussel performance. Further to this, mussels in duplicate systems were inoculated with Escherichia coli and Vibrio parahaemolyticus to inform on risks associated with pathogen proliferation. The mussels interacted with the seawater in both the RSW and Periodic RSW systems as seawater ammonia concentrations increased in both. Mussel mortalities counted across the 10-day trials were highest in the Chilled treatment (5.5%), followed by RSW (3.5%) and Periodic RSW (2.4%). Key measurements including haemolymph pH and osmolality, meat and intra-valvular water weight, and textural hardness, were all affected by the post-harvest treatments. For inoculated mussels, E. coli and V. parahaemolyticus most probable numbers (MPN) remained stable in both the Chilled and Periodic RSW treatments with reductions in both bacteria recorded in the RSW treatment. Higher variances in intra-valvular water retention and haemolymph haemocyte numbers and differences in volatile organic compound profiles were seen in the summer mussels, as compared with spring, highlighting mussel condition as a determining factor for post-harvest performance. RSW has therefore been demonstrated to be beneficial, but optimisation (e.g., through further reduction of metabolic activity) may further increase practical post-harvest transport distances and the quality of live/fresh mussel products.

EPS9.07 Improving colistin activity against Pseudomonas aeruginosa biofilms

Colistin is the last-resort therapeutic option in infections by multi-drug resistant pathogens and for this it has been included by World Health Organization among the “highest priority critically important antimicrobials.” Colistin binding to outer membrane (OM) of Gram negative bacteria leads to membrane destabilization, increasing permeability and finally cell death. OM remodeling, by chemical groups that reduce its negative charge, induces resistance to colistin. In Pseudomonas aeruginosa addition of aminoarabinosyl residues (Ara4N) to the lipid-A is the major pathway leading to colistin resistance, whose last step is catalyzed by the aminoarabinosyl transferase ArnT. We have previously identified a natural compound, BBN149 (renamed FDO), as putative inhibitors of ArnT, which potentiates colistin activity against planktonic colistin-resistant P aeruginosa strain (Ghirga et al., 2020, doi:10.1093/jac/dkaa200). As biofilms are relevant to the persistence of bacterial infection, we tested the colistin adjuvant activity of FDO, and its derivative FDO-H, on P. aeruginosabiofilms. First, biofilms were grown in medium containing colistin, and the resulting biofilms have been quantified by crystal violet, metabolic assay and viable cell counting. The resulting data showed a dose-dependent reduction in metabolic activity, total biomass and cell viability of biofilms treated with colistin. In biofilms treated with colistin and FDO or FDO-H, viable cells counting showed the adjuvant activity of both compounds with one log reduction of CFU in sample treated with colistin +compound respect to colistin only. Importantly, similar results were obtained with an in vitro evolved P. aeruginosa reference strain (PA14 colR5, doi:10.1128/AAC.01820-17) and with a clinical colR isolate. Collectively, our data suggest that FDO and FDO-H potentiate colistin activity against P. aeruginosa biofilms.

Anti-Candidal Activity of Reboxetine and Sertraline Antidepressants: Effects on Pre-Formed Biofilms.

Reboxetine (REB) and sertraline (SER) are antidepressants. The antifungal potential of these drugs against planktonic Candida has been recently reported with limited data about their effects on Candidal biofilms. Biofilms are self-derived extracellular matrixes produced by the microbial population that is attached to biotic surfaces, such as vaginal and oral mucosa, or abiotic surfaces, such as biomedical devices, resulting in persistent fungal infections. The commonly prescribed antifungals, azoles, are usually less effective when biofilms are formed, and most of the prescribed antifungals are only fungistatic. Therefore, the current study investigates the antifungal potentials of REB and SER, alone and in combination with fluconazole (FLC) and itraconazole (ITR) against Candidal biofilms. Using proper controls, Candida species (Candida albicans, C. albicans; Candida krusei, C. krusei; and Candida glabrata, C. glabrata) were used to form biofilms in 96-well microplates. Serial dilutions corresponding to concentrations ranging from 2 to 4096 µg/mL of the target drugs (REB, SER, FLC, ITR) were prepared and added to the plates. Impairment of the biofilm biomass and biofilm metabolic viability was detected using the crystal violet (CV) assay and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, respectively. In the checkerboard assay, the sessile fractional inhibitory concentration index (SFICI) was calculated to evaluate the effects of drug combinations. SER was more effective in reducing the biomass than REB for C. albicans and C. glabrata, but both were equal for C. krusei. For the reduction in metabolic activity in C. albicans and C. glabrata, SER had a slight advantage over REB. In C. krusei, REB was slightly more potent. Overall, FLC and ITR were almost equal and produced more significant reductions in metabolic activity when compared to SER and REB, except for C. glabrata, where SER was almost equal to FLC. Synergism was detected between REB + FLC and REB + ITR against biofilm cells of C. albicans. Synergism was detected between REB + ITR against biofilm cells of C. krusei. Synergism was detected between REB + FLC and REB + ITR against biofilm cells of C. albicans, C. krusei, and C. glabrata. The results of the present study support the potential of SER and REB as anti-Candidal biofilm agents that are beneficial as a new antifungal to combat Candidal resistance.