Applied Biosciences

Integrated crop and livestock systems (ICLSs) have been considered an important management-based decision to improve soil health by carbon sequestration. A two-year study (2019–2021) at CPBES in Newton, MS, was conducted to evaluate the effect of an ICLS on soil microbial diversity in the southeastern region of the USA, representing agroclimatic conditions that are warm and humid. Amplicons targeting bacterial 16S rRNA genes and fungal ITS2 regions were sequenced. Taxonomic assignment and characterization of microbial diversity were performed using QIIME2^®. Soil fungal diversity pattern showed significant difference (alpha diversity, p = 0.031 in 2020 and beta diversity, p = 0.037 in 2021). In contrast, no significant differences were observed in bacterial diversity. However, there were several beneficial bacterial phyla, such as Proteobacteria and Actinobacteria, and fungal phyla such as Ascomycota, which were dominant in both years and did not show significant differences due to cover crop treatments. Canonical Correspondence Analysis (CCA) and Mantel test showed significant influence on fungal diversity due to carbon (rm = 0.2581, p = 0.022), nitrogen (rm = 0.2921, p = 0.0165), and electrical conductivity (rm = 0.1836, p = 0.0583) in 2021, and on bacterial diversity due to EE-GRSP (rm = 0.22, p = 0.02) in 2020. However, the results showed that there were no significant differences between the cover crop treatments that were consistent over a two-year study period. However, the mix of different cover crops such as oats (Avena sativa L.), crimson clover (Trifolium incarnatum L.), and tillage radish (Raphanus sativus L.) demonstrated higher positive correlation and lower negative correlation with different bacterial and fungal phyla. Long term study of ICLS is suggested to understand the shift in microbiome that would help in understanding the role of cover crops and grazing in improving crop production sustainably. Full article

This study explores the interaction between Vemurafenib (VEM), a potent BRAF inhibitor, and calf thymus DNA (ctDNA) using a comprehensive array of biophysical and computational techniques. The primary objective is to understand the potential off-target effects of VEM on DNA, given its established role in melanoma therapy targeting the BRAF V600E mutation. The investigation employed methods such as ultraviolet–visible absorption spectroscopy, steady-state fluorescence, circular dichroism, isothermal titration calorimetry, and advanced molecular dynamics simulations. The results indicate that VEM interacts with DNA primarily through a minor groove-binding mechanism, causing minimal structural disruption to the DNA double helix. Viscosity measurements and melting temperature analyses further confirmed this non-intercalative mode of binding. Calorimetry data revealed an exothermic, thermodynamically favorable interaction between VEM and ctDNA, driven by both enthalpic and entropic factors. Finally, computer simulations identified the most probable binding site and mode of VEM within the minor groove of the nucleic acid, providing a molecular basis for the experimental findings. Full article

Osteoarthritis is a joint disease that causes pain, stiffness, and reduced joint function because the protective cushioning inside the joints, called cartilage, gradually wears away. This condition is caused by various factors and complex processes in the joint’s environment, involving different types of cells producing factors that can either maintain the joint health or contribute to osteoarthritis. This study aimed to understand the factors influencing both healthy and diseased joints in DDD strategies for the in vitro preconditioning of MSCs. An electronic search in the PubMed, Scopus, and Web of Science databases was carried out using the terms (cartilage OR chondr*) AND (repair OR regeneration OR healing) AND (niche OR microenvironment)) AND (“growth factor” OR GF OR cytokine). Researchers used various methods, including macroscopic examinations, histology, immunohistochemistry, and microCT. Molecules associated with joint inflammation were identified, like macrophage markers, MMP-13, TNF, apoptotic markers, and interleukins. Chondrogenesis-related factors such as aggrecan GAG, collagen type II, and TGF beta family were also identified. This study suggests that balancing certain molecules and ensuring the survival of joint chondrocytes could be crucial in improving the condition of osteoarthritic joints, emphasizing the importance of chondrocyte survival and activity. Future preconditioning methods for MSC- and EV-based therapies can find suitable strategies in the described microenvironments to explore co-culture systems and soluble or extracellular matrix factors. Full article

Metallo-β-lactamases (MBLs) are members of the structurally conserved but functionally diverse MBL-fold superfamily of metallohydrolases. MBLs are a major concern for global health care as they efficiently inactivate β-lactam antibiotics, including the “last-resort” carbapenems, and no clinically suitable inhibitors are currently available. Increasingly, promiscuous β-lactamase activity is also observed in other members of the superfamily, including from viruses, which represents an underexplored reservoir for future pathways to antibiotic resistance. Here, two such MBL-fold enzymes from Bacillus phages, the cyclic mononucleotide-degrading proteins Apyc_Goe3 and Apyc_Grass, are shown to degrade β-lactam substrates efficiently in vitro. In particular, Apyc_Grass displays a distinct preference for carbapenem substrates with a catalytic efficiency that is within one order of magnitude of the clinically relevant MBL NDM-1. Mutagenesis experiments also demonstrate that the loss of a metal-bridging aspartate residue reduces nuclease activity up to 35-fold but improves carbapenemase activity. In addition, we hypothesise that the oligomeric state significantly influences β-lactamase activity by modifying access to the active site pocket. Together, these observations hint at a possible new avenue of resistance via the spread of phage-borne MBL-fold enzymes with β-lactamase activity. Full article

Papaya is a globally important crop, with production primarily based on hermaphrodite plants. Papaya has three sex types—male, female, and hermaphrodite—determined by flower morphology, but this is only distinguishable at the flowering stage. In this study, a recombinase polymerase amplification (RPA) assay was developed and optimized to identify the three sexes of papaya. Recombinant uvsX, uvsY, gp32, and Bsu DNA polymerase were used to study the effects of temperature, reaction time, and sensitivity conditions for RPA reaction efficiency. The optimal conditions were found to be 41 °C and a 30 min reaction time, allowing the detection of the target sex from specific DNA markers, even when using crude extract. This study shows that RPA could be used for sex determination in papaya, and the findings could contribute to developing a point-of-need strategy due to their sensitivity and specificity. Full article

The olive tree is currently cultivated worldwide, with higher incidence in Mediterranean Basin. Its climate is highly favorable to the synthesis of phenolic compounds, stored in olive leaves; their consumption has been linked to a lower incidence of cancer and cardiovascular disorders for which the research interest upon this feature has increased in last decade. This study aimed (i) to review evidence about the importance of olive leaf extract (OLE) on human health and the physiological effect of its major compounds; (ii) to update the state of the art of studies conducted on the health and technological usage of olive leaf extract; (iii) to report potential uses of OLE in pharmaceuticals, food production, and cosmetics; and (iv) to prospect the future of clinical applications of OLE from diverse cultivars, especially in metabolic inflammatory conditions such as polycystic ovary syndrome (PCOS). Overall, cultivars richer in TPC, including TFC, αT, omega-3 and omega-9, present a main research target for supplementation alone or in conjunction with vitaminic compounds, due to their nutraceutical value in metabolic disorders, chronic inflammatory diseases, and anti-aging treatments, whereas cultivars with less water content might be useful as substrates for food preservation. With regard to future prospects, it would be of great interest to clarify the specific mechanisms underlying the beneficial effects of OLE on neuro-immune and cardiovascular health to design safer and healthier nature-based medicine for a wide array of costly and highly prevalent chronic diseases, such as inflammatory and metabolic-related syndromes, namely, PCOS. Full article

Grape pomace is a winery byproduct that is rich in polyphenols with antioxidant capacity. This study investigated the effect of 0, 5, and 10% inclusion of dehydrated grape pomace (DGP) in finishing pig diets on the growth performance, carcass traits, fatty acid profile, fresh meat quality, and fat stability of a local pig genotype. A total of 36 pigs, 18 barrows, and 18 gilts (83.23 ± 6.03 kg and 132.1 ± 5.6 days old) were allotted in a randomized block design considering the initial weight (block) within sex, with six replicates of each sex per treatment. Including DGP in the diets did not affect daily weight gain or the feed-to-gain ratio; however, daily feed intake increased linearly (p < 0.05) and backfat thickness at the last rib, backfat thickness at the first sacral vertebrae, P2 backfat thickness, fat area, and the percentage of lean meat decreased linearly (p < 0.05) in pigs. The inclusion of DGP in pig diets did not affect the antioxidant potential evaluated by thiobarbituric acid-reactive substances in mini hamburgers or the quality characteristics of fresh meat, except for intramuscular fat (EE). The dietary inclusion of DGP linearly increased (p < 0.05) EE, saturated fatty acids, monounsaturated fatty acids, and Σω-3 and reduced the ω-6:ω-3 ratio in a linear way (p < 0.05) in the loin of pigs. We concluded that it is feasible to include up to 10% of DGP in pig diets without affecting growth performance, but carcass quality may be impaired due to increased adiposity. Furthermore, meat quality can be improved by increasing intramuscular fat and ω-3 fatty acid content, but fat stability is not affected when DGP is included at up to 10% of the diet for 49 days prior to slaughter. Full article

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