Applied Biosciences

Protoplasts offer a promising alternative to embryogenic cell suspensions (ECS) for gene editing in banana, potentially overcoming several limitations associated with ECS-based transformation systems. This study aimed to optimize protoplast isolation and regeneration in Cavendish banana (cv. Williams) and to assess their suitability for transient gene expression. Enzymatic digestion of ECS using cellulase and macerozyme consistently yielded approximately 3 × 10⁶ protoplasts per milliliter of settled cell volume. Protoplast yield was further enhanced, by approximately threefold, through the addition of an antioxidant mixture (ascorbic acid, citric acid and L-cysteine) combined with 0.01% bovine serum albumin. Polyethylene glycol-mediated transfection with a green fluorescent protein reporter gene yielded transient expression in approximately 0.75% of protoplasts five days post-transfection. While phenotypically normal plants were regenerated from untransfected protoplasts after 12 weeks in agarose bead culture with conditioned liquid medium, no regeneration was observed from transfected cells. These findings establish a reproducible protocol for protoplast isolation and plant regeneration in Cavendish banana and provide insight into the barriers limiting successful regeneration following transfection. Full article

Improving the efficacy of microbial biocontrol agents is a pivotal strategy for sustainable management of rice blast and sheath blight caused by Pyricularia oryzae and Rhizoctonia solani, respectively, in Vietnam. In this study, Trichoderma sp. TVN-A0 and Trichoderma sp. TVN-H0 were irradiated by gamma to generate mutants for screening the enhanced antagonistic activity against P. oryzae and R. solani. The potential mutants were screened by antifungal metabolite production via the cellophane membrane assay (I_CM), antagonistic performance through dual culture confrontation assays (I_DC), volatile organic compound bioassays (I_VOCs), and chitinase activity. As a result, among five potential mutants derived from each wild-type strain (AM1-AM5 and HM1-HM5), mutant AM2 originated from TVN-A0, and mutant HM2 derived from TVN-H0 demonstrated the highest inhibition rates and chitinase activities. The AM2 exhibited I_CM of 96.71% against R. solani, 92.57% against P. oryzae, I_DC of 87.76%, and I_VOCs of 83.57%, while HM2 possessed I_CM of 95.33% against R. solani, 85.28% against P. oryzae, I_DC of 91.24%, and I_VOCs of 79.33%. The genetic differences among mutants and their parents were investigated by RAPD. The non-GMO AM2 and HM2 mutants are promising candidates for biocontrol of the diseases caused by P. oryzae and R. solani in Vietnam. Full article

Deformed wing virus (DWV) is a major contributor to honey bee colony losses, making effective monitoring essential for apiary management. Traditional DWV detection relies on hazardous RNA extraction followed by RT-qPCR, creating barriers for widespread surveillance. We developed an immunocapture RT-qPCR (IC-RT-PCR) method for screening DWV-A infections by capturing intact virus particles from bee homogenates using immobilized antibodies. Validation demonstrated strong correlation with TRIzol^®-based extraction (r = 0.821), with approximately 6 Ct reduced sensitivity, consistent with other published immunocapture methods. Performance was adequate for moderate–high viral loads, while TRIzol^® showed superior detection for low-dose infections. Laboratory-produced reverse transcriptase showed equivalent performance to commercial enzymes, providing cost savings. IC-RT-PCR eliminates hazardous chemicals and offers a streamlined workflow for surveillance screening where the safety and cost benefits outweigh the sensitivity reduction. This method provides a practical alternative for large-scale DWV-A surveillance programs, while TRIzol^® remains preferable for low-level detection and diagnostic confirmation. Full article

The present research features an experimental comparative design and the objective of this work was to determine the susceptibility to microbiological contamination in fatty acid methyl esters (FAMEs) and the FAME–water interface of residual and refined lard, large volume simulating storage conditions as fuel supply chain, and to identify the microorganisms developed. The plates were seeded according to ASTM E-1259 and the instructions provided by the manufacturer of the Bushnell Haas agar. Microbiological growth was observed at the FAME–water interface of FAME obtained from residual lard. Using the MALDI-TOF mass spectrometry technique, Pseudomonas aeruginosa and Streptomyces violaceoruber bacteria were identified in the residual lard FAMEs, with the latter being previously reported in FAMEs. The implications of microorganism development on the physicochemical quality of FAMEs are significant, as it leads to an increase in the acid index, which may negatively impact metals by inducing corrosion. The refined lard FAMEs did not show any development of microorganisms. The present research concluded that residual lard tends to be more prone to microbiological attack if the conditions of water and temperature affect microbial growth. The findings will contribute to the knowledge base for a safer introduction of FAMEs into the biofuel matrix. Full article

Toothpaste is an essential oral hygiene product commonly used to sustain oral health due to its incorporation of antimicrobial agents. Numerous functional toothpastes enriched with antimicrobial agents have been developed and are available to consumers. This study evaluates the antimicrobial and antibiofilm efficacy of 12 commercially available toothpaste products, including those with specialized functions. Statistical significance was assessed to validate the differences observed among the toothpaste samples. Their effects on Streptococcus mutans, the primary pathogen responsible for dental caries, were evaluated. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined, and bacterial growth was measured to compare antimicrobial activities. Toothpaste containing 1000 μg/mL fluoride and whitening toothpaste exhibited the strongest antimicrobial effects, effectively inhibiting S. mutans growth. Additionally, bamboo salt-enriched and tartar-control toothpaste demonstrated inhibitory effects on bacterial growth. Assays to evaluate the ability of cells to form biofilms and the expression of genes involved in biofilm formation revealed a partial correlation between biofilm formation and spaP, gtfB, gtfC, and gtfD expression, although some showed opposite trends. Collectively, this study provides valuable insights into the antimicrobial and biofilm inhibition capabilities of commercial toothpastes against S. mutans, offering a foundation for evaluating the efficacy of functional toothpaste products. Full article

Plant latex is a rich source of proteolytic enzymes with potential biomedical applications, particularly in hemostasis. Among them, thrombin-like enzymes (TLEs) have garnered interest in their ability to mimic thrombin by catalyzing the conversion of fibrinogen to fibrin, facilitating clot formation. While TLEs from snake venoms have been well-characterized and applied clinically, their plant-derived counterparts remain underexplored. This review critically examines the structural and functional characteristics of TLEs from plant latex, comparing them to animal-derived TLEs and evaluating their role in both procoagulant and fibrinolytic processes. Emphasis is placed on dual fibrinogenolytic and fibrinolytic activities exhibited by latex proteases, which often vary with concentration, incubation time, and protease type. In vitro coagulation assays and electrophoretic analyses are discussed as critical tools for characterizing their multifunctionality. By addressing the knowledge gaps and proposing future directions, this paper positions plant latex proteases as promising candidates for development in localized hemostatic and thrombolytic therapies. Full article

The discovery that human beings may endogenously produce ethanol is not new and dates back at the end of the 19th century; recently, however, it has become clear that through the proliferation of gut microorganisms that produce ethanol from sugars or other substrates, blood alcohol level may be greater than 0, despite Homo sapiens sapiens lacking the enzymatic pathways to produce it. Very rarely this can lead to symptoms and/or to a disease, named gut fermentation syndrome or auto-brewery syndrome (ABS). The list of microorganisms (mostly bacteria and fungi) is very long and contains almost 100 different strains, and many metabolic pathways are involved. Endogenous ethanol production is a neglected entity, but it may be suspected in patients in whom ethanol consumption may be firmly excluded. Nevertheless, due to the growing prevalence of NAFLD (now renamed as MAFLD) worldwide, an ethanol-producing microorganism responsible for endogenous ethanol production such as Klebsiella pneumoniae or Saccharomices cerevisiae is increasingly sought in NAFLD patients, or in patients with metabolic diseases such as diabetes mellitus, obesity, or metabolic syndrome, at least in selected instances. In the absence of standard diagnostic and therapeutic guidelines, ABS requires a detailed patient history, including dietary habits, alcohol consumption, and gastrointestinal symptoms, and a comprehensive physical examination to detect unexplained ethanol intoxication. It has been proposed to start the diagnostic protocol with a standardized carbohydrate challenge test, followed, if positive, by the use of antifungal agents or antibiotics; indeed, fecal microbiota transplantation might be the only way to cure a patient with refractory ABS. Scientific societies should produce internationally agreed recommendations for ABS and other conditions linked to excessive endogenous ethanol production. Full article

Phyllosphere microbial communities influence the growth and productivity of plants, particularly in epiphytic plants, which are disconnected from nutrients available in the soil. We characterized the phyllosphere of 30 individuals of the epiphytic cycad, Zamia pseudoparasitica, collected from three forest sites during the rainy and dry seasons in the Republic of Panama. We used DNA metabarcoding to describe the total bacteria community with the 16S rRNA gene and the diazotrophic community with nifH gene. Common taxa included members of the Rhizobiales, Frankiales, Pseudonocardiales, Acetobacteriales, and the diazotrophic community was dominated by Cyanobacateria. We observed similar patterns of alpha diversity across sites and seasons, and no community differences were seen within sites between the rainy and dry seasons for either the 16S rRNA or nifH genes. However, pairwise comparisons showed some statistically significant differences in community composition between sites and seasons, but these explained only a small portion of the variation. Beta diversity partitioning indicated that communities were more phylogenetically closely related than expected by chance, indicative of strong environmental or host filtering shaping these phyllosphere communities. These results highlight the influence of host-driven selection and habitat stability in shaping phyllosphere microbiota, offering new insights into microbial assembly in tropical canopy ecosystems. Full article

Recent findings have identified high-density lipoprotein (HDL) as a carrier of microRNAs, small non-coding RNAs that regulate gene expression, suggesting a potential novel functional and biochemical role for HDL-microRNA cargo. Here, we conduct an in-depth bioinformatics analysis of unique HDL-microRNA cargo to uncover their molecular mechanisms and potential applications as clinical biomarkers. First, using the Gene Expression Omnibus (GEO), we performed computational analysis on public human microRNA array datasets (GSE 25425; platform GPL11162) obtained from highly purified fractions of HDL in human plasma in order to identify their unique miRNA cargo. This led to the identification of eleven miRNAs present only in HDL, herein listed: hsa-miR-210, hsa-miR-26a-1, hsa-miR-628-3p, hsa-miR-31, hsa-miR-501-5p, hsa-miR-100-3p, hsa-miR-571, hsa-miR-100-5p, hsa-miR-23a, hsa-miR-550, and hsa-miR-432. Then, these unique miRNAs present in HDL were analyzed using a bioinformatics approach to recognize their validated target genes. The ClusterProfiler R package applied gene ontology and KEGG enrichment analysis. The key genes mainly enriched in the biological process of cellular regulation were identified and linked to neurodegeneration. Finally, the protein–protein interaction and co-expression network were analyzed using the STRING and GeneMANIA Cytoscape plugins. Full article

Robinia pseudoacacia is one of the most widely introduced—but also controversial—tree species in Europe. On the one hand, it is valued for its productivity, timber quality, and melliferous blossom. On the other hand, it is highly invasive and causes habitat change and homogenization. The aim of the study reported on here was to assess the genetic diversity of selected R. pseudoacacia stands in Lithuania in districts with the highest black locust stands frequency and to evaluate its spatial distribution in self-establishing stands. To achieve this aim, we employed four nuclear SSR loci (Rops 02, Rops 05, Rops 06, and Rops 08) and investigated the genetic diversity of five R. pseudoacacia plots. The study results reveal that R. pseudoacacia in Lithuania is genetically diverse (the average allele number per plot was 3.66, and the average Ho was 0.83). R. pseudoacacia in the plots forms tight clonal groups that hardly intermix with each other; it also spreads by seeds (66 single-copy genotypes were found in total in all 5 investigated plots). R. pseudoacacia stands in Lithuania originate from different seed sources and from different introduction events, as revealed by the allelic pattern, genetic diversity, and genetic differentiation among the research plots. Full article

The intensification of olive growing has raised environmental concerns, particularly regarding nutrient loss from excessive fertiliser use. In line with the European Union’s Farm to Fork strategy, which aims to halve the soil nutrient losses by 2030, this study evaluates the effectiveness of two sensor-based approaches—proximal sensing with a FLAME spectrometer and remote sensing via UAV-mounted multispectral imaging—compared with foliar chemical analyses as the reference standard, for diagnosing the nutritional status of olive trees. The research was conducted in Elvas, Portugal, between 2022 and 2023, across three olive cultivars (‘Azeiteira’, ‘Arbequina’, and ‘Koroneiki’) subjected to different fertilisation regimes. Machine learning (ML) models showed strong correlations between sensor data and nutrient levels: the multispectral sensor performed best for phosphorus (P) (determination coefficient [$R^2$] = 0.75) and potassium (K) ($R^2$ = 0.73), while the FLAME spectrometer was more accurate for nitrogen (N) ($R^2$ = 0.64). These findings underscore the potential of sensor-based technologies for non-destructive, real-time nutrient monitoring, with each sensor offering specific strengths depending on the target nutrient. This work contributes to more sustainable and data-driven fertilisation strategies in precision agriculture. Full article

Jaboticaba berry is a rich source of polyphenols with bioactive properties. However, polyphenols are known for their high reactivity under environmental conditions, which poses a challenge to producing stable, functional components for the food industry. This study investigated the storage stability and bioaccessibility of polyphenols in microencapsulated jaboticaba juice over 21 days at three storage temperatures: −20 °C, 4 °C, and 25 °C. Additionally, phenolic compounds and antioxidant capacity were evaluated before and after in vitro simulated gastrointestinal digestion. Microencapsulation was performed by spray drying at 160 °C using maltodextrin at different concentrations (10%, 12%, and 15%) as the wall material. The results showed that the stability of polyphenols during storage was significantly influenced by both temperature and the proportion of maltodextrin. Greater degradation of phenolic compounds was observed at 25 °C, particularly in the formulation with 10% maltodextrin. On the other hand, the bioaccessibility of polyphenols was significantly higher in microencapsulated juice after simulated gastrointestinal digestion compared to non-encapsulated jaboticaba juice (p < 0.05). These findings suggest that microencapsulation technique improved the bioaccessibility of phenolic compounds in jaboticaba and promoted better stability with the use of a higher concentration of maltodextrin. In conclusion, microencapsulation is a promising strategy for the development of functional food products enriched with natural bioactive compounds, providing greater protection and efficiency in delivering their health benefits. Full article

In this paper, we present three log-linear models for comparing spectral density functions (SDFs) of neural spike sequences (NSSs). The logarithmic (ln) ratios of the estimated SDFs are modeled as polynomial expressions with respect to angular frequencies plus residual series with autocorrelated errors. The advantage of the proposed models is that they can be applied within certain frequency ranges. Analysis of point processes in the frequency domain can be performed to obtain estimates of the SDFs of NSSs by smoothing the mean-corrected periodograms using moving average weighting schemes. The weighting schemes may differ in the estimated SDFs. To decorrelate the error terms in the log models, we apply a two-stage method: in the first stage, the error terms are identified by choosing a suitable model, while in the second stage, the reliable estimates of the unknown parameters involved in the polynomial expressions are derived by decorrelating the data. An illustrative example from the field of neurophysiology is described, in which the neuromuscular system of the muscle spindle is affected by three different stimuli: (a) a gamma motoneuron, (b) an alpha motoneuron, and (c) a combination of gamma and alpha motoneurons. It is shown that the effect of the gamma motoneuron on the muscle spindle is shifted by the presence of the alpha motoneuron to lower frequencies in the range of [1.03, 7.6] Hz, whereas the presence of the gamma motoneuron shifts the effect of the alpha motoneuron in two bands of frequencies: one in the range of [13.5, 19.9) Hz and the other in the range of [19.9, 30.8] Hz. Full article

Linear DNA constructs are used in gene delivery and therapy application due to their capacity of integration into the mammalian genome, offering stable transgene expression. Compared to circular plasmids, linear DNA also has the advantage that its dimension and steric hindrance are directly correlated to the length of the nucleotide chain. These considerations make linear DNA an effective choice for gene delivery pilot studies, where formulations and transfection efficiency calculations are studied considering the nucleic acid dimensions. Meanwhile, the development of DNA–chitosan nanoparticles (NPs) has gained significant interest for their potential in nucleic acid delivery, especially as non-viral gene delivery systems and for embedding linear DNA fragments, as well as gene delivery to the lung. This study explored an easy polyelectrolyte complexing preparation of linear DNA-loaded chitosan nanoparticles. Among the different formulations of nanoparticles prepared, the optimal one exhibited a size of approximately 290 nm, an encapsulation efficiency of 86% and a zeta potential of 25 mV. Additionally, this study examined how the concentration of DNA in solution influenced nanoparticle formation, encapsulation efficiency and particle size. In particular, transient transfection of the chitosan–linear DNA fragment complex, encoding for green fluorescent protein (GFP), was conducted in human pulmonary distal lung cells (NCI-H441 cells), demonstrating successful cellular internalization and protein expression. These studies highlight the potential of DNA–chitosan NPs in nucleic acid delivery, particularly for pulmonary applications. Future works will focus on formulating the achieved carrier into an inhalable dosage form to improve its translational application. Full article

Flow cytometry is widely applied to infer the ploidy and genome size (GS) of plant nuclei. The conventional approach of sample preparation, reliant on fresh plant material to release intact nuclei, often results in poor yields of nuclei in conditions when a plant material cannot be kept fresh due to logistical constraints. Previous attempts to use frozen plant material were mainly limited to ploidy analysis and relied on chopping methods, which restrict the material input and often result in poor nuclei yield, especially in frozen samples, due to incomplete disruption. Here, we present a modified protocol for GS estimation using frozen plant material that facilitates larger volumes of tissue to be processed while improving debris removal. Nuclei isolated from this protocol can also be used for DNA or RNA extraction. Genome size estimates from frozen material are similar to those from fresh material, with a reduction in error range, although not always significant (p > 0.05). In certain species, frozen samples can yield substantially more nuclei than fresh material. With the addition of specific debris compensation algorithms, coefficient of variation (CV%) can be maintained below 5%. This method has special value in estimating the GS of samples collected from remote locations and frozen for use in plant genome sequencing. Freezing preserves high-quality DNA and RNA, enabling the same sample to be used for both flow cytometry and genome sequencing. Full article

An aqueous leaf extract of Egeria densa was used to green-synthesize iron (II) and iron (III) oxide nanoparticles from ferrous sulphate and ferric chloride, respectively. The successful green synthesis of the nanoparticles was confirmed through UV–visible spectroscopy, and the colour of the mixtures changed from light-yellow to green-black and reddish-brown for FeO–NPs and Fe₂O₃–NPs, respectively. The morphological characteristics of the nanoparticles were determined using an X-ray diffractometer (XRD), a Fourier transform infrared spectrophotometer (FTIR), a transmission electron microscope (TEM), and energy-dispersive X-ray spectroscopy (EDX). The UV–Vis spectrum of the FeO–NPs showed a sharp peak at 290 nm due to the surface plasmon resonance, while that of the Fe₂O₃–NPs showed a sharp peak at 300 nm. TEM analysis revealed that the FeO–NPs were oval to hexagonal in shape and were clustered together with an average size of 18.49 nm, while the Fe₂O₃-NPs were also oval to hexagonal in shape, but some were irregularly shaped, and they clustered together with an average size of 27.96 nm. EDX analysis showed the presence of elemental iron and oxygen in both types of nanoparticles, indicating that these nanoparticles were essentially present in oxide form. The XRD patterns of both the FeO–NPs and Fe₂O₃–NPs depicted that the nanoparticles produced were crystalline in nature and exhibited the rhombohedral crystal structure of hematite. The FT-IR spectra revealed that phenolic compounds were present on the surface of the nanoparticles and were responsible for reducing the iron salts into FeO–NPs and Fe₂O₃–NPs. Conclusively, this work demonstrated for the first time the ability of Elodea aqueous extract to synthesize iron-based nanoparticles from both iron (II) and iron (III) salts, highlighting its versatility as a green reducing and stabilizing agent. The dual-path synthesis approach provides new insights into the influence of the precursor oxidation state on nanoparticle formation, thereby expanding our understanding of plant-mediated nanoparticle production and offering a sustainable route for the fabrication of diverse iron oxide nanostructures. Furthermore, it provides a simple, cost-effective, and environmentally friendly method for the synthesis of the FeO–NPs and Fe₂O₃–NPs using Egeria densa. Full article

This review examines the history of consumption, life cycle, and culture conditions of seven edible mucilaginous terrestrial cyanobacterial strains—Nostoc flagelliforme, Nostoc commune, Nostoc sphaeroides, Nostoc sphaericum, Nostoc verrucosum, Aphanothece sacrum, and Nostochopsis lobatus—as resilient and sustainable food sources in the face of climate change. Traditionally consumed across various cultures and known for their resilience in extreme environments, these cyanobacteria offer high nutritional value, including proteins, vitamins, and essential fatty acids, making them promising candidates for addressing food security. Their ability to fix nitrogen reduces reliance on synthetic fertilizers, enhancing agricultural applications by improving soil fertility and minimizing dependence on fossil fuel-derived chemicals. Unlike conventional crops, these cyanobacteria require minimal resources and do not compete for arable land, positioning them as ideal candidates for low-impact food production. Despite these advantages, the review highlights the need for scalable and cost-effective cultivation methods to fully realize their potential in supporting a resilient global food supply. Additionally, it underscores the importance of ensuring their safety for consumption, particularly regarding toxin content. Full article

Carnitine palmitoyltransferase 1 (CPT1), which catalyzes the rate-limiting step of fatty acid oxidation, has been implicated in therapeutic approaches to several human diseases characterized by aberrant lipid metabolism. The isoform-specific quantification of CPT1 activity is essential in the characterization of small molecule inhibitors of CPT1, but several existing means to quantify enzymatic activity, including the use of radioisotope-labeled carnitine, are not amenable to scalable, high throughput screening. Here, we demonstrate that mitochondrial extracts from Expi293 cells transfected with a CPT1a plasmid are a reliable and robust source of catalytically active human CPT1. Moreover, with a source of catalytically active enzyme in hand, we modified a previously reported colorimetric method of coenzyme A (CoA) easily scalable to a 96-well format for the screening of CPT1a inhibitors. This assay platform was validated by two previously reported inhibitors of CPT1a: R-etomoxir and perhexiline. To further demonstrate the applicability of this method in small molecule screening, we prepared and screened a library of 87 known small molecule APIs, validating the inhibitory effect of chlorpromazine on CPT1. Full article

Prostate cancer (PCa) is one of the most common malignancies in men, where early and accurate detection is crucial. While PSA testing has been the diagnostic standard, its limited specificity leads to unnecessary biopsies and missed significant cancers. Urinary biomarkers such as PCA3 and TMPRSS2-ERG and multi-marker assays (MyProstateScore, SelectMDx, and ExoDx) offer a promising alternative. This narrative review examines their diagnostic performance and clinical utility with the aim of understanding whether they can be integrated with the established tests and exams already in use. A literature search of PubMed, Scopus, and Medline identified some relevant recent studies (2010–2025). The findings show that PCA3 and TMPRSS2-ERG improve specificity over PSA, while multi-marker tests enhance risk stratification and reduce unnecessary procedures. MPS integrates urinary biomarkers with PSA, achieving over 95% sensitivity and negative predictive value for clinically significant cancers. SelectMDx demonstrates ~90% negative predictive value, and ExoDx assesses urinary exosomes to predict aggressive disease. Despite their advantages, challenges persist, including variability in performance, cost, and accessibility. Urinary biomarkers represent a major step toward more precise, less invasive diagnostics, with future research needed to optimize clinical integration and cost-effectiveness. Full article