Applied Biosciences

The commercial value of kiwifruit is determined mostly by its nutritional composition and antioxidant content. The enrichment of these traits in an era where climate change affects kiwi growth conditions is worth investigating via the application of biostimulants that enhance plant growth. In this work, we evaluated the effects of three commercial biostimulants on the metabolism and growth characteristics of the Actinidia deliciosa cultivar ‘Hayward’: (I) a humic and fulvic-based biostimulant, (II) a glycine–betaine–proline-based biostimulant, and (III) a vegetal amino acid-based biostimulant. A biostimulant-free treatment was used as a control. In the two-year experiment (2022 and 2023), the growth characteristics of kiwi trees were measured, such as stem length, the number of leaves, leaf area, and fresh and dry biomass at the end of each growing season. The leaves’ total chlorophyll, total phenolics, and proline content were detected during the two seasons in order to obtain more insights into plant metabolism. Κiwifruit qualities and antioxidant traits, such as total soluble solids, titratable acidity, firmness, fresh and dry biomass, DPPH, and ascorbic acid (vitamin C), were recorded during the postharvest life for each season. Data analysis illustrated the beneficial impact of some biostimulants on several of the previously mentioned parameters, such as antioxidant kiwifruit traits, especially in the case of glycine–betaine–proline-based acid-based biostimulants. Full article

Stool is a mixture of excrement, microbiota, enzymes, undigested material, and small molecules. Fecal metabolomics has gained interest recently, owing to advances in metabolomics and growing research into both the host’s physiology and the gut microbiome. One challenge with fecal metabolomics is preserving the sample integrity from collection until analysis, as the microbiota and enzymes continue to alter the metabolome following defecation. Currently, flash-freezing or lyophilization are utilized to minimize post-collection metabolome changes; however, this requires complex equipment and immediate processing, precluding the possibility for at-home sampling. Commercial devices containing stabilizing solvents have been developed to facilitate at-home collection, ambient transport, and sample storage. Here, we explore the efficacy of a commercially available stool collection device with a stabilization reagent tailored to fecal metabolomics. Stool samples from six donors were either processed shortly post-collection or stored at room temperature for seven days in the tube, with and without the stabilization reagent. Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS)-based untargeted metabolomics was utilized for analyzing extracted metabolites. Chemometric analysis was used to evaluate the performance of the device. We found that the device with the stabilization reagent minimized changes in the metabolite profile relative to unstabilized stool left at room temperature for one week. Full article

Escherichia coli serotype O157:H7 is a diarrheal agent and a significant cause of hemorrhagic colitis and the development of hemolytic uremic syndrome (HUS), mainly in infants. Early detection of contaminated food and water using reliable and fast tests is one of the strategies to prevent infections from E. coli O157:H7. Methods: Four quantitative polymerase chain reaction protocols (SYBR Green qPCR) were developed and validated to determine the presence of the bacteria according to its rfbE, stx₁, and stx₂ genes. Results: The results of the efficiencies were between 80% and 97% with a high linearity (R² 0.99). The cut-off limits for each primer sequence were 3.1667 × 10⁻² ng µL⁻¹ for two sequences of the serogroup O157 (primers rfbE and O157), 1.7228 × 10⁻³ ng µL⁻¹ for stx₁, and 3.5185 × 10⁻³ ng µL⁻¹ for stx₂. The inclusivity and the exclusivity of each gene, as well as the analytical precision and the positive and negative predictive value, were 100%. A contaminated meat matrix was evaluated, detecting up to 4 CFU g⁻¹. Conclusions: SYBR Green qPCR protocols could be implemented to trace the presence of E. coli O157 in a routine analysis of ground beef or as an easy, rapid, sensitive, and specific diagnostic test while still considering microbiological tests to validate any inconclusive results. Full article

The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), causes significant damage to olive crops worldwide. However, the factors affecting its survival under water deprivation have not been studied yet. In this study, the water deprivation resistance of male and female olive fruit flies was measured at three ages in virgin and mated adults fed either a full or a restricted diet. The experiments (24 treatments) were conducted under constant laboratory conditions, using insects collected in the wild and reared on olives. Additionally, a baseline experiment was conducted to provide data on the insects’ life expectancy under no-stress conditions. Our findings revealed that males showed much less resistance under water deprivation compared to females. Younger adults endured for longer than older ones, and adults fed a restricted diet endured water deprivation longer than those fed a full diet. Our results suggest that during periods of water scarcity, releasing sterile males is most effective, because the wild male population decreases. Since females of reproductive age are more resistant, this should ensure a higher number of matings with sterile males. These findings can be used to formulate improved pest control strategies that enhance olive product quality while relying less on insecticides. Full article

As isobutanol exhibits higher energy density and lower hygroscopicity than ethanol, it is considered a better candidate biofuel. The sustainable supply of inedible biomass and lack of competition with the food supply have stimulated significant worldwide interest in the production of isobutanol from this resource. Both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) have been applied to isobutanol production to effectively utilize inedible biomass as a feedstock. However, both processes have various challenges, including low isobutanol yield and high production costs. This review summarizes the potential of isobutanol as a biofuel, methods for conferring isobutanol productivity, recent experimental studies, and developments in both SHF and SSF with the isobutanol-producing strains. Challenges to increasing the isobutanol yield and various suggestions for improvements to enable commercial production are also discussed. Full article

The nucleus accumbens shell (NAcSh) is a major structure associated with distinct aspects of reward and mnemonic information encoding, relying on spatial data to define optimal behavioral strategies. Chronic pain-derived striatal plasticity is considered one underpinning cause of working memory (WM) impairments. However, it is unclear how the NAcSh is involved in these spatial deficits. To address this, we evaluated the impact of unilateral local NAcSh electrical lesions during the execution of a food-reinforced eight-shaped spatial alternation WM task. Behavioral performance was assessed in rats after the onset of the neuropathic pain model—spared nerve injury (SNI). Our findings indicate that the induction of SNI and/or NAcSh lesions did not significantly impact the animals’ performance accuracy or motor activity during the execution of the behavioral task, but altered their response latency patterns. In addition, these manipulations did not induce significant antinociceptive effects. Collectively, these results suggest that the NAcSh may participate in specific aspects of spatial information integration and processing under neuropathic pain conditions. Full article

Pulse proteins are playing significant roles in the alternative protein space due to the demand for foods produced in an environmentally sustainable manner and, most importantly, due to the demand for foods of nutritious value. There has been extensive research to mimic animal-derived meat texture, flavour, mouthfeel, etc. However, there is still the perception that many of the plant-based proteins that have been texturized to mimic meat are still highly processed and contain chemicals or preservatives, reducing their appeal as being healthy and precluding any sustainable benefits. To counter this notion, the biotransformation of pulse proteins using enzymes or fermentation offers unique opportunities. Thus, this review will address the significance of pulse proteins in the alternative protein space and some of the processing aids leading to the isolation and modification of such protein concentrates in a sustainable manner. Fermentation-based valorization of pulse proteins will also be discussed as a “clean label” strategy (further adding to sustainable nutritious plant protein production), although some of the processes like the extensive use of water in submerged fermentation need to be addressed. Full article

Thermoplastics used to construct a variety of patient medical devices can become contaminated by harmful bacteria. We investigated whether two different Bacillus species recovered from patient radiation therapy thermoplastic masks could similarly contaminate thermoplastic material used to construct patient orthoses (splints). Bacillus bacteria form dormant spores, which have been shown to enhance its attachment to thermoplastics. Bacterial attachment and recovery were examined using an orthotic thermoplastic with an anti-stick coating being compared to uncoated material used in radiation therapy applications. Triplicate sample squares were seeded with a saline suspension of either B. cereus (MAB03F) or B. megaterium (DAB01F) containing a similar number of spores. Squares were subsequently sampled at 1 h, 1 week, 2 weeks, 4 weeks, and 8 weeks. The number of recovered bacteria was counted. Differences in material hydrophobicity were determined by water contact angle analysis. Both Bacillus species attached to each material within 1 h, and their spores were recovered at 8 weeks. However, a decreasing trend in adhesion, over time, was noted to the coated material with an opposite increasing trend in the uncoated material. Decreased Bacillus species spore adhesion to coated material with a lower hydrophobicity suggests a greater potential for spore transfer to patients wearing contaminated orthoses. Full article

Synucleinopathies, typified by Parkinson’s disease (PD), entail the accumulation of α-synuclein (αSyn) aggregates in nerve cells. Various αSyn mutants, including the αSyn A53T variant linked to early-onset PD, increase the propensity for αSyn aggregate formation. In addition to disrupting protein homeostasis and inducing proteostatic stress, the aggregation of αSyn in PD is associated with an imbalance in iron metabolism, which increases the generation of reactive oxygen species and causes oxidative stress. This study explored the impact of αSyn A53T expression in transgenic hairy roots of four medicinal plants (Lobelia cardinalis, Artemisia annua, Salvia miltiorrhiza, and Polygonum multiflorum). In all tested plants, αSyn A53T expression triggered proteotoxic stress and perturbed iron homeostasis, mirroring the molecular profile observed in human and animal nerve cells. In addition to the common eukaryotic defense mechanisms against proteostatic and oxidative stresses, a plant stress response generally includes the biosynthesis of a diverse set of protective secondary metabolites. Therefore, the hairy root cultures expressing αSyn A53T offer a platform for identifying secondary metabolites that can ameliorate the effects of αSyn, thereby aiding in the development of possible PD treatments and/or treatments of synucleinopathies. Full article

Chitinases are biotechnologically relevant enzymes that can be applied in such different sectors as pharmaceutical, food, environmental management, the biocontrol of pests and in the paper and cellulose industry. Microorganisms as filamentous fungi are the most important source of these biomolecules. The fungus Aspergillus niveus produces extracellular chitinase when cultured under submerged fermentation using shrimp shells, a residue generated by the fish industry, as a carbon source, for 96 h at 30 °C and 100 rpm. The particle size and concentration of the shrimp shells affected enzyme production. The chitinase was purified until electrophoretic homogeneity through the use of a Sephadex G-100 chromatographic column. It is a monomeric glycoprotein with a molecular mass of 47 kDa estimated using SDS-PAGE and 49.3 kDa determined using gel filtration. The carbohydrate content was 22.8%. The best temperature and pH for enzyme activity were 65 °C and 6.0, respectively. Approximately 80% of the enzymatic activity was preserved at pH 4.0 and 5.0 for 48 h, and the half-life (t₅₀) was maintained for 48 h at 40 °C. Salts, EDTA and β-mercaptoethanol did not affect chitinase activity significantly, but organic solvents reduced it. The kinetic parameters determined using p-NPGlycNac were K_m of 2.67 mmol L⁻¹, V_max of 12.58 U mg of protein⁻¹, Kcat of 2.47 s⁻¹ and K cat/K_m of 0.93 s⁻¹ mmol L⁻¹. The A. niveus chitinase inhibited the growth of all fungal strains used, especially Trichoderma harzianum (MIC = 22.4 μg mL⁻¹) and Penicillium purpurogenum (MIC = 11.2 μg mL⁻¹). The chitinase produced by A. niveus presented interesting characteristics that indicate its potential of application in different areas. Full article

The utilization of microalgae as a green carbon source for chemical production has attracted attention for its potential use in sustainable and climate-friendly solutions. This study investigates the growth of Dunaliella salina, a unicellular green microalga, in response to salinity variations and water and seawater addition to compensate for evaporation in open cultures. The impact of continuous and non-continuous water addition, as well as seawater addition, on the growth of D. salina was analyzed though tank tests. The results showed that different water-addition methods did not significantly influence cell concentrations, indicating the organism’s resilience to salinity changes. Continuous water addition maintained stable salinity levels at 12%, but required continuous monitoring, while non-continuous addition reduced the intervention frequency. The overall results showed that a salinity range between 12 and 15% did not affect microalgae growth, suggesting flexibility in evaporation-loss compensation methods based on cultivation-system specifics and resource availability. Maintaining consistent biomass regardless of the water-addition method used suggests sustainable production within the tested salinity range, with seawater addition making microalgae cultivation more adaptable to regions with varying water availability. Further research, including outdoor pilot tests, is recommended to validate and extend these findings to natural environments. Full article

Growing demands for sustainable and ecological nanoparticle synthesis methods have incentivized the scientific community to develop new approaches to counteract these challenges. Green synthesis resorts to biocomponents obtained from plants, bacteria, fungi, and other organisms to synthesize nanostructures, with beneficial gains in the economic and ecological cost associated with the process, simplicity of the process, and resource efficiency. Moringa oleifera, a native plant originally from India with immense nutritive value, has long been used by researchers in the biosynthesis of nanoparticles. Leaves, flowers, bark, and seeds are among the “miracle tree” parts that can be used in nanoparticle green synthesis. Moringa oleifera seed cake, a by-product obtained from defatted seeds, is often overlooked due to its apparent low commercial value. The main objective of this review is to highlight the recent findings reported in the literature on nanoparticles/nanocomposites synthesized with seed cake biocompounds acting as reducing/capping agents. Furthermore, we analyzed the methods currently employed for the extraction of bioactive compounds. Moringa oleifera seed for industrial applications was also addressed. Full article

The emergence of bacteria resistant to bacteriophage (phage) infection may compromise the success and effectiveness of phage therapy. The aim of this study was to evaluate the in vitro antibacterial activity of five novel phages, as well as the emergence of bacterial resistance to phage infections. The antibacterial activity of lytic phages was evaluated against standard strains of Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25927), Enterococcus faecalis (ATCC 29212) and Staphylococcus aureus (ATCC 6538). Phages were initially grown in the presence of host bacteria in an exponential growth phase, then purified and titrated. In a second exposure, 20 μL of each phage was inoculated with 10⁶ CFU/mL of P. aeruginosa/E. coli/E. faecalis/S. aureus, separately. In a third exposure, resistant colonies were isolated, cultivated and exposed again to the phages. Bacterial colonies resistant to phage infection after the third exposure were evaluated for their susceptibility profile to different antibiotics via the diffusion disk technique. The diameters of the inhibition halos were evaluated with Image J software (version 1.54g) and the definition of the susceptibility profile to antibiotics was determined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. In addition, fourteen cocktails with different phages were formulated to evaluate the emergence of a bacterial resistance to phage infections. The phages exhibited specificity for P. aeruginosa and did not infect E. coli, E. faecalis and S. aureus. The presence of bacterial colonies resistant to phage infection in the three successive exposures was identified, and the bacterial resistance to phage infection was confirmed in all phages titrated at 10⁸ PFU/mL, in four phages titrated at 10¹⁰ PFU/mL and in one phage titrated at 10¹³ PFU/mL. The development of a resistance to infection by phages (~10⁸ PFU/mL) did not change the susceptibility profile of P. aeruginosa to antibiotics and, when evaluating the emergence of a resistance to infection by phage cocktails (~10⁸ PFU/mL, ~10¹⁰ PFU/mL, ~10¹³ PFU/mL), bacterial resistance to phage infection was confirmed in all cocktails with phages titrated at 10⁸ PFU/mL, in ten cocktails with phages titrated at 10¹⁰ PFU/mL and in seven cocktails with phages titrated at 10¹³ PFU/mL. In conclusion, the presence of resistant P. aeruginosa colonies to phage infection after successive exposures was evidenced, although some phages at title ~10¹⁰ PFU/mL and ~10¹³ PFU/mL were effective in inhibiting the growth of resistant colonies. The development of resistance did not change the susceptibility profile of P. aeruginosa to antibiotics. Variants of P. aeruginosa that were resistant to phage infection were isolated and their resistance to infection via the phage cocktail was demonstrated regardless of the viral titer, although some cocktails at title ~10¹⁰ PFU/mL and ~10¹³ PFU/mL were effective in inhibiting the growth of resistant colonies. Despite the emergence of bacterial variants resistant to phage infection, new studies involving the applicability of phages in the control of infections must be conducted. Full article

Introduction: Mortality from acute radiation syndrome is frequently caused by hematopoietic or gastrointestinal radiotoxicity, the latter of which currently has no effective treatment. Transforming growth factor-beta 3 (TGF-β3) may decrease the severity of radiation-induced gastrointestinal damage in mice. In addition, treatment with TGF-β3 may alleviate radiation-induced fibrosis. Objectives: The current study aimed to investigate the effect of TGF-β3 treatment on acute and late radiotoxicity in whole body irradiated mice. Methods: C57BL/6J mice were total body irradiated with 8.5 Gy X-rays with or without shielding of one hind leg to alleviate hematopoietic radiotoxicity. The effects of intravenous TGF-β3 treatment were investigated. Body weight and pain expression were monitored. Intestine, lung, and liver tissues were preserved and analyzed. Alpha smooth muscle actin (α-SMA) expression in MRC-5 cells after 3.5 Gy X-irradiation combined with TGF-β3 treatment was analyzed using flow cytometry. Results: All total body irradiated animals died within ten days after irradiation. Ninety-three percent of femur-shielded mice survived until sampling or termination. No effect of TGF-β3 treatment was observed in either group. No increase in collagen content was detected in the lungs or liver from irradiated mice regardless of TGF-β3 treatment. In vitro, α-SMA expression increased synergistically after irradiation and TGF-β3 treatment. Conclusions: Shielding of the femur during total body irradiation decreased acute gastrointestinal radiation toxicity and increased survival. TGF-β3 treatment did not impact symptoms or survival. TGF-β3 treatment and irradiation increased α-SMA expression in MRC-5 cells synergistically. Full article

As climate change is an imminent threat to the environment and agriculture, there is an increasing need to find immediate solutions capable of compensating for water deficits even in semi-arid conditions. This study is focused on the evaluation of the vegetative growth of grapevine plants Vitis vinifera L., of the Greek variety “Debina” in a water deficit environment, with the application of two bacterial-based formulations: one with Bacillus amyloliquefaciens (strain QST 713) and one with Sinorhizobium meliloti (strain cepa B2352). The two formulations were tested under rational irrigation (100% of Available Water) and deficit irrigation (57% of AW). After 140 days, plant growth parameters, such as total plant growth length, leaf area, roots, shoots, and leaves dry biomass showed better performance on treatments with plant growth-promoting rhizobacteria (PGPR) formulations under either rational or deficit irrigation conditions. In addition, the metabolic response of the grapevine plants to the deficit irrigation stress, such as the total chlorophyll, leaf relative water, total phenolic, and proline content, proved to be enriched on the treatments with PGPR formulations during this experiment. The two formulations, in conditions of abiotic stress, achieved to almost compensate for the irrigation deficit, boosting the plant metabolism. This study reveals the need for further research on PGPR biostimulants, as this first trial of these formulations on grapevine could be significant in the case of water scarcity and climate change. Full article

Flavonoids share a common structural framework that serves as a hallmark indicative of their biological activity. In this study, we investigated the effects of two structurally similar flavonoids, fisetin and morin, through independent and combined in vitro assessments on embryonic mouse cells overexpressing the human 70 kDa heat shock protein (Hsp70) (Tg/Tg) and normal mouse fibroblast cell line (NIH/3T3). The primary objectives were to evaluate the biocompatibility and potential cytotoxicity of these flavonoids, along with assessing the cytoprotective role of Hsp70 in these cellular environments. To address these objectives, we conducted dose- and time-dependent cell survival tests. Additionally, we utilized flow cytometry to detect intracellular reactive oxygen species (ROS) production and to analyze apoptosis and the cell cycle. Throughout the experimental procedures, a notable observation was made: NIH/3T3 normal cells exhibited greater susceptibility compared to Tg/Tg cells when exposed to fisetin and morin. This difference in susceptibility is likely attributed to the robust cytoprotective effect of Hsp70 in Tg/Tg cells. Importantly, both cell lines demonstrated increased sensitivity to fisetin toxicity in comparison to morin, leading to significantly lower cell survival rates. These findings shed light on the differential responses of cell lines to flavonoid exposure, emphasizing the influence of Hsp70 and the distinct impact of fisetin and morin on cell viability. Full article

Background and objectives: Carbohydrates such as fructooligosaccharides (FOSs) are associated with improved gastrointestinal health and the prevention of excess body fat. We evaluated the long-term effects of high amounts of FOS on metabolic parameters, non-alcoholic fatty liver disease (NAFLD) and short-chain fatty acids (SCFAs). Methods: Sixty C57BL/6 mice received the following diets for four months: control (C), normolipid rich in fiber (F), normolipid supplemented with FOS (FOS), high fat (HL), high fat with high fiber (HLF) and high fat with FOS (HLFOS). We analyzed the animal weight; body composition; food intake; fasting blood glucose; serum and liver lipid profiles; liver and intestinal histologies; malondialdehyde (MDA), hepatic retinol and α-tocopherol; and SCFAs in the feces. Results: Supplementation with FOS in a high-fat diet promoted less body weight gain and reduced liver and retroperitoneal adipose tissue weights compared to HL and HF. FOS prevented NASH and decreased alanine aminotransferase and serum cholesterol levels in experimental animal models of obesity and metabolic syndrome (MS). There were statistical differences found in the dosages of the three main SCFAs in feces (acetic, isobutyric and isovaleric acids). Conclusions: Long-term supplementation with high doses of FOS was effective in reducing weight, adiposity, NAFLD and serum cholesterol in C57BL mice with obesity and MS induced by a high-fat diet. Full article

Torn and damaged menisci resulting from trauma are very common knee injuries, which can cause pain and mobility limitations and lead to osteoarthritis. Meniscal injuries can require surgery to repair the tissue damage and restore mobility. Here we describe the biomechanical testing of a 3D-printed meniscus to illustrate methods to determine if it has the strength and durability to effectively repair meniscal tears and restore knee biomechanics. This work was designed to demonstrate the steps needed to test novel meniscus repair devices prior to moving toward animal testing. The first testing step determined the ability of the 3D-printed meniscus to withstand surgical fixation by measuring the suture pull-out force. We show that vertical 2/0 silk or Fiberwire sutures need an average of 1.4 or 1.8 N, respectively, to pull through the meniscus, while horizontal sutures need only 0.7 and 1.2 N, respectively. The next step measured the compressive strength of normal, damaged, and repaired porcine meniscus tissue. Here, we show that meniscectomy decreased the stiffness of meniscus tissue from 26.7 ± 0.85 N to 7.43 ± 0.81 N at 25% strain. Menisci repaired with the 3D-printed tissue restored 66% of the measured force at 25% strain. The final step measured the contact pressures and areas in an ex vivo porcine knee before and after meniscal repair was made with the 3D-printed meniscus tissue. The example 3D-printed meniscus was successfully sutured into the porcine knee joint but failed to restore normal knee contact pressures. This work demonstrates the need for an iterative biomechanical testing process of biomaterial development, 3D-printing optimization, and knee kinematics to develop a durable and functional meniscus repair device. In summary, the methods described here serve as a guide for the functional evaluation of novel meniscus repair devices. Full article

Consumers are looking for safer and more natural food options that are produced through natural methods without using synthetic preservatives. They also desire extended shelf life for their food products. Several medicinal and aromatic plants species combine food, spice, aromatic, and medicinal recognized attributes. The essential oils from these plants contain a unique mixture of compounds specific to each plant, showing notable antioxidant and antimicrobial properties. Essential oils are used widely as they are environmentally friendly, non-toxic, and biodegradable substitutes for harsh chemical preservatives. Thyme and clove are aromatic plants commonly used in traditional gastronomy, particularly in meat-based recipes. The preservation effects of these essential oils on fresh meat have not been widely studied. Therefore, the aim of this study is to review the use of thyme and clove essential oils in meat preservation, with particular emphasis on their antioxidant properties to mitigate lipid and protein oxidation. Different strategies have been used to boost the effects of essential oils in foods, which include mixtures of essential oils, encapsulation and nanoemulsification techniques, with or without edible coatings. The final objective is to promote the wide use of essential oils for meat preservation, eventually in combination with other innovative approaches. Full article