Applied Biosciences doi: 10.3390/applbiosci5040098

Authors: María Jesús Ramírez-Expósito José Manuel Martínez-Martos Cristina Cueto-Ureña

Physiological brain aging is driven by progressive neuro-glial-vascular remodeling, in which neurotransmitter systems serve as the core functional network integrating central resilience and peripheral systemic signaling. This narrative review provides a comprehensive synthesis of the molecular, cellular, and circuit-level mechanisms underlying healthy brain aging. Centered on classical and modulatory neurotransmitter systems, we examine how neurochemical trajectories interact with key regulators of cellular resilience, including the SIRT1/Klotho/mTORC1 signaling network, epigenetic modifications such as the N(6)-methyldeoxyadenosine (N6medA) clock, extracellular matrix dynamics, and multi-organ (muscle–gut–liver–brain) axes. We also discuss how socioeconomic and environmental exposures, such as childhood poverty and air pollution, contribute to individual aging trajectories throughout life. Recent advances in multimodal neuroimaging, spatial transcriptomics (IRISeq), and AI-based brain-age estimation (e.g., ResNet-CrossViT) are highlighted as promising approaches for identifying biomarkers capable of distinguishing physiological aging from the earliest stages of neurodegeneration. Collectively, healthy brain aging represents a dynamic process of biological adaptation in which neurotransmitter remodeling, cellular resilience, metabolic homeostasis, and systemic interactions jointly determine cognitive outcomes. Understanding these interconnected mechanisms provides a framework for developing preventive strategies aimed at preserving cognitive function and extending brain health span.

Applied Biosciences doi: 10.3390/applbiosci5040097

Authors: Patrícia Sesterheim Fernando da Silva Martins Fernanda Marques da Silva Eduardo Rolim Teixeira

Background: Extracellular vesicle (EV)-associated microRNAs (miRNAs) have been implicated in intercellular signaling processes relevant to bone regeneration; however, the regulatory relationships through which selected EV-associated miRNAs may converge on biological processes relevant to bone repair remain incompletely understood. Objective: To investigate, using an in silico systems biology approach, the predicted regulatory architecture of five literature-selected EV-associated miRNAs potentially involved in biological processes relevant to bone regeneration. Methods: Five miRNAs (hsa-miR-146a-5p, hsa-miR-21-5p, hsa-miR-335-5p, hsa-miR-503-5p, and hsa-miR-129-5p) were selected a priori based on published evidence linking them to EV-associated signaling and/or osteogenesis-related biological processes. Candidate target genes were predicted using TargetScan Human 8.0 and prioritized according to Context++ score ranking, followed by predicted target-overlap analysis, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment pathway enrichment, and miRNA–messenger RNA (mRNA) network reconstruction using Cytoscape 3v.10. Results: The five miRNAs yielded 395 prioritized predicted miRNA–mRNA interactions corresponding to 387 unique predicted target genes, with most predicted targets associated with a single miRNA and a limited subset shared between pairs of miRNAs. Eight genes were identified as pairwise shared predicted targets, with no higher-order overlap involving three or more miRNAs. SMAD family member 7 (SMAD7) was identified as was identified as a pairwise shared predicted target of hsa-miR-21-5p and hsa-miR-503-5p and was selected for biological contextualization because of its involvement in transforming growth factor beta (TGF-β)/bone morphogenetic protein (BMP)-associated signaling. Functional enrichment identified statistical overrepresentation of signaling pathways regulating pluripotency of stem cells, as well as mitogen-activated protein kinase (MAPK), Hippo, TGF-β, Wnt, mechanistic target of rapamycin (mTOR), and forkhead box O (FoxO) signaling pathways. Conclusions: This exclusively computational analysis identified a predicted regulatory architecture characterized by predominantly miRNA-specific target relationships, limited pairwise target overlap, and pathway-level enrichment potentially relevant to bone-related biological processes. These findings are hypothesis-generating and do not establish miRNA abundance within a specific EV preparation, direct miRNA–mRNA regulation, pathway activation, or osteoregenerative activity. Experimental EV cargo characterization and functional validation in relevant cellular and in vivo models are required to determine the biological significance of the predicted relationships.

Applied Biosciences doi: 10.3390/applbiosci5040096

Authors: Ricardo Santillán-Mendoza Nelly A. González-Oviedo Mauricio Andrade-Marcial Berenice Preza-Murrieta Humberto J. Estrella-Maldonado Felipe R. Flores-de la Rosa

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), severely impairs photosynthesis and chloroplast function in citrus. However, Persian lime (Citrus latifolia) exhibits remarkable tolerance, maintaining productivity despite infection. Pentatricopeptide repeat (PPR) proteins regulate organellar RNA metabolism and are essential for chloroplast function. Here, we investigated whether plastid-targeted PPR regulatory networks are transcriptionally altered during CLas infection. We combined transcriptome-wide identification of PPR proteins, prediction of chloroplast RNA targets, gene expression analyses, and chlorophyll a fluorescence measurements. A total of 515 PPR proteins were identified, with 30.5% predicted to localize to plastids. Predicted RNA targets included chloroplast genes involved in transcription and photosynthesis, such as ycf2, rpoC1, psbB, and psbC. Despite this extensive predicted regulatory network, neither plastid-targeted PPR genes nor their chloroplast targets showed differential expression between HLB-positive and HLB-negative leaves. In contrast, infected leaves exhibited reduced Fv/Fm, PIabs, and electron transport efficiency, although the characteristic OJIP transient remained largely preserved, indicating maintenance of PSII structural organization. These findings suggest that constitutive plastid PPR regulatory mechanisms remain stable during CLas infection and may contribute to the chloroplast resilience underlying HLB tolerance in Persian lime.

Applied Biosciences doi: 10.3390/applbiosci5040095

Authors: Sander Bekeschus Hiromasa Tanaka Vandana Miller

The 11th International Conference on Plasma Medicine (ICPM11), held jointly with the 10th International Workshop on Plasmas for Cancer Treatment (IWPCT10), took place from 31 May to 5 June 2026 in Gifu, Japan. The meeting brought together 225 scientists from 23 countries across four continents, reflecting the continued growth and interdisciplinary nature of plasma medicine. The scientific program included 24 invited lectures, 51 contributed oral presentations, and 133 poster presentations, covering advances in plasma–biological interactions, plasma-based cancer therapy, wound healing, antimicrobial applications, plasma-activated liquids, sterilization, and emerging biomedical and agricultural applications. This conference report provides an overview of ICPM11 and summarizes the scientific contributions presented during the meeting. The breadth of topics highlights the rapid progress of plasma medicine from fundamental research toward clinical and technological applications, emphasizing the importance of international collaboration in advancing the field.

Applied Biosciences doi: 10.3390/applbiosci5040094

Authors: Emily Fichter Johanna Bellstedt Henriette Bretschneider Jörg Lützner Anne Bernhardt

The poor regenerative capacity of articular cartilage and the limited efficacy of current treatments in restoring its extracellular matrix contribute to osteoarthritis. This study investigated an emerging approach using the secretome of mesenchymal stromal cells (MSC) to stimulate chondrocyte-driven cartilage matrix synthesis. MSC from human bone marrow (hBM-MSC) and Wharton’s jelly (hWJ-MSC) were cultured in various 3D culture systems, including spheroids, alginate beads, porous collagen scaffolds and bilayer collagen membranes in vitro. Conditioned medium (CM) was collected and analyzed for anti-inflammatory and trophic factors via ELISA. Furthermore, the CM was applied to human chondrocytes (hCh), which were assessed for chondrogenic and osteogenic gene expression and histologically stained for type II collagen (COL II) and sulfated glycosaminoglycans (sGAG). hCh cultured in chondrogenic differentiation medium served as a control. MSC remained viable in all four application models. Growth differentiation factor 5 (GDF-5) release was significantly increased for hWJ-MSC compared to hBM-MSC. Anti-inflammatory factors remained undetectable in the present setup. CM of 3D cultivated MSC differently affected chondrogenic differentiation of chondrocyte pellets, with CM of spheroids showing higher effects compared to the other three application models. This study demonstrates that the secretion of cytokines by MSC is strongly affected by the microenvironment of the cells.

Applied Biosciences doi: 10.3390/applbiosci5040093

Authors: Norma Elvira Muguruza Crispin Carmen del Pilar Alvarez Quinteros Genaro Christian Pesantes Arriola Elvira Teófila Castañeda Chirre Fiorella Victoria Luperdi Rios Jacqueline Roxana Reaño Rivera Marleni Vilma Bautista Espinoza Franklin O. Areche Olivia Magaly Luque Vilca Gisella Patty Lacherre Rodriguez Lucy Emilia Torres Carrera Bertha Milagros Villalobos Meneses William Andres Guzmán Sánchez Eufemio Magno Macedo Barrera Geraldina Rebeca Parihuaman Quinde

Fish-processing by-products represent a potential source of peptide-based plant biostimulants, but the relationships between peptide molecular characteristics and plant metabolic responses remain poorly defined. This study evaluated the effects of peptide molecular-weight fraction (<500, 500–1500, and 1500–3000 Da), activation method (crude biol, enzymatic hydrolysis, and microbial fermentation), and foliar application timing (vegetative, rosette, and preharvest) on purple lettuce (Lactuca sativa L.) under open-field conditions. Peptide characterization was integrated with physiological, carbon–nitrogen metabolic, anthocyanin, and transcriptional analyses. Enzymatic activation substantially increased hydrolysis and functional characteristics within each molecular-weight class; for example, apparent degree of hydrolysis increased by 80.4% in S2 relative to S1, while ORAC increased by 52.5%. Foliar treatments were associated with marked physiological differences, with S2C3 showing a 65.4% lower contact angle and 36.6% higher net photosynthetic rate than S7C1. Across the selected carbon–nitrogen metabolic contrasts, nitrate reductase and glutamine synthetase activities increased by 383.9% and 422.6%, respectively, while total soluble sugars and total free amino acids increased by 146.0% and 202.4%. Anthocyanin-related enzyme activities and transcript abundance also differed among treatments. Total quantified anthocyanins increased from 50.9 ± 3.1 mg kg−1 FW in S9C1 to 127.0 ± 4.6 mg kg−1 FW in S1C3, representing a 149.5% increase. Overall, lower-molecular-weight activated peptide preparations were associated with stronger physiological and metabolic responses, although responses varied among individual endpoints. These findings support molecular characterization and application timing as important considerations in developing fish-derived peptide biostimulants while providing a route for valorizing fish-processing by-products.

Applied Biosciences doi: 10.3390/applbiosci5040092

Authors: Teresa Maria Rosaria Regina Loretana Angela De Maldè Valentina Femia

Authentication of commercial oregano products remains challenging because processing may hinder the identification of undeclared botanical species. In this study, a stepwise molecular approach integrating DNA mini-barcoding, species-specific real-time PCR, and loop-mediated isothermal amplification (LAMP) was developed for the detection of undeclared botanical components in commercial oregano products. Eighty-five commercial oregano products were initially screened by DNA mini-barcoding targeting the plastidial trnL region. Sequence analysis identified Myrtus communis, Olea europaea, and Convolvulus althaeoides among the detected botanical species. Based on the identification of M. communis, species-specific real-time PCR and LAMP assays were developed and applied for its targeted detection, for which a species-specific molecular assay in oregano products has not previously been reported. M. communis was detected in 31 samples by real-time PCR and in 42 samples by LAMP, whereas 18 samples were identified as positive by DNA mini-barcoding among the subset subjected to cloning and sequencing. Sample-level comparison showed partial concordance among the three approaches, supporting their complementary use for M. communis detection. The LAMP assay showed specific amplification of M. communis, with a detection limit of 1 pg of target genomic DNA and consistent detection down to a 0.01% matrix-spiking level in oregano tissue. Overall, the proposed approach combines broad DNA-based screening with targeted molecular detection, while the newly developed trnL-based LAMP assay provides a rapid tool for the detection of undeclared M. communis in processed oregano products.

Applied Biosciences doi: 10.3390/applbiosci5040091

Authors: Konstantinos Liaras Giovanni N. Roviello Christos Petrou Yiannis Sarigiannis

The increasing incidence of fungal infections caused by Candida albicans and the multidrug-resistant emerging pathogen Candidozyma auris (formerly Candida auris) represents a major global healthcare challenge. Current antifungal therapies are limited by toxicity, drug–drug interactions, rapid resistance emergence, and poor efficacy against fungal biofilms, highlighting the urgent need for novel antifungal agents with new mechanisms of action. In this context, marine ecosystems have emerged as an exceptional source of structurally diverse natural products with promising pharmacological properties. This review summarizes recent advances in marine-derived compounds exhibiting antifungal activity against C. albicans and C. auris. The available evidence is organized according to major chemical classes, including polyketides, alkaloids, terpenoids and meroterpenoids, and miscellaneous marine metabolites. Particular emphasis is placed on antifungal potency, mechanisms of action, anti-biofilm and anti-virulence activities, synergistic interactions with conventional antifungal drugs, and reported structure–activity relationships. Several compounds demonstrated potent direct antifungal activity, while others showed mainly antibiofilm, anti-virulence, synergistic, or resistance-modifying effects despite having limited direct growth-inhibitory activity. Although many promising candidates remain at the preclinical stage, current evidence suggests that marine natural products have significant potential as scaffolds for discovering and developing next-generation antifungal therapeutics. Continued interdisciplinary research integrating natural product chemistry, medicinal chemistry, pharmacology, and translational studies will be essential for transforming these compounds into clinically useful antifungal drugs.

Applied Biosciences doi: 10.3390/applbiosci5040090

Authors: Kapka Mancheva Georgi Atanasov

Single-pulse transcranial magnetic stimulation (spTMS) is widely used to investigate human corticospinal and cortical physiology, yet individual physiological measures provide only partial information about cortical function. This systematic review aimed to synthesize evidence on human physiological responses evoked by spTMS, with particular emphasis on resting and active motor thresholds, motor-evoked potentials (MEPs), corticospinal silent periods (CSPs), and biological and technical determinants of response variability. The review was conducted in accordance with PRISMA 2020 and searched PubMed, Scopus, Web of Science, and Google Scholar, supplemented by citation tracking. Evidence was synthesized qualitatively because of substantial heterogeneity in stimulation protocols, populations, recording methods, and outcomes. Across the included literature, no single measure fully characterized corticospinal excitability: motor thresholds primarily reflected membrane/axonal excitability, MEPs reflected integrated corticospinal output, and CSPs provided information on intracortical inhibitory processes. Age, sex-related anatomical and hormonal factors, cortical anatomy, coil position and orientation, pulse waveform, stimulation intensity, muscle state, attention, fatigue, sleep, and medication contributed to response variability. The findings support an integrated interpretation of spTMS measures, standardized protocols, and individualized electric field modeling. TMS-evoked EEG potentials (TEPs) methodology is discussed only as complementary contextual information; TEPs were not systematically targeted by the original search strategy and were therefore not included among the outcomes of the systematic synthesis. The exact historical search date could not be reconstructed from the available documentation and is reported transparently as a limitation.

Applied Biosciences doi: 10.3390/applbiosci5040089

Authors: Cătălina Raluca Nuță Lidia Băjenaru Ana Maria Moiceanu Sovarel Ovidiu Lucian Băjenaru

Cognitive frailty combines physical frailty with cognitive impairment without dementia, identifying older adults vulnerable to adverse outcomes. Candidate microbiome-related pathways in cognitive frailty include dysfunction of the intestinal epithelial and blood–brain barriers, altered microbial metabolites, systemic and vascular inflammation, anabolic resistance, and neuroinflammation. However, direct evidence is scarce, and most findings derive from physical frailty, sarcopenia, mild cognitive impairment, or Alzheimer’s disease, inviting overinterpretation. This review integrates a multi-database evidence map with a domain-based synthesis of phenotypes, mechanisms, biomarkers, confounding, interventions, and clinical translation. Conducted up to 31 July 2026, the search identified one eligible primary human study directly examining the gut microbiome in cognitive frailty. Its small sample, age and educational imbalances, 16S-based resolution, and lack of external validation preclude clinical biomarker claims despite apparent discrimination. Larger metagenomic frailty studies report lower microbial diversity, gene richness, and butyrate-production capacity, with partial cross-population replication. Dietary, prebiotic, and probiotic trials demonstrate microbiome target engagement and report selected physical or cognitive benefits, but these outcomes are inconsistent, rarely assessed together, and not established as microbiome-mediated; fecal microbiota transplantation remains experimental. The microbiome should be considered a potential component of multidomain risk models rather than a stand-alone biomarker. Translation requires standardized phenotyping, longitudinal multi-omics, causal diagrams, external validation, and trials with concurrent physical and cognitive endpoints.

Applied Biosciences doi: 10.3390/applbiosci5040088

Authors: Monjurul Haq Sharmin Suraiya Raju Ahmed Md. Tanvir Ahmed Md. Sadek Ali Vikash Chandra Roy Jin-Seok Park Hee-Jeong Lee Truc Cong Ho Ratih Pangestuti Man Seog Chun Byung-Soo Chun

Hilsa muscle biomass was hydrolyzed to recover bioactive proteins and peptides using subcritical water hydrolysis (SWH) with ultrasound pretreatment at various temperatures (150–250 °C) and pressures (40–80 bar). Hydrolysis yield was increased with rising temperature and pressure, with the highest yield (98.70 ± 0.60%) observed at 250 °C and 80 bar using SWH with ultrasound pretreatment (UPT). The properties of the hydrolysates—including pH, taurine content, and color values—were influenced by the hydrolysis conditions. Additionally, total protein content, reducing sugar levels, antioxidant activity, foaming capacity (FC), and foaming stability (FS) increased at higher temperatures and pressures. Hydrolysates prepared at 225 °C and 70 bar, as well as at 250 °C and 80 bar, contained highly bioactive peptides and molecules that exhibited inhibitory effects on hydroxyl radicals and prevented λ DNA degradation. The highest 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) activities were observed in hydrolysates obtained at 225 °C and 70 bar with ultrasound pretreatment, reaching 96.88 ± 0.26% and 76.36 ± 2.84%, respectively. Amino acid profile analysis showed that the greatest number of amino acids (eight) was detected in hydrolysates produced through SWH at 250 °C and 80 bar. Overall, SWH combined with ultrasound pretreatment markedly enhanced yield and biofunctional properties of hilsa protein hydrolysates, highlighting their potential applications in food, pharmaceutical, and cosmetic industries.

Applied Biosciences doi: 10.3390/applbiosci5040087

Authors: Joisleen Ramírez Josué Prens Zohre Kurt Arthur Mc Carty James

Water contamination by heavy metals is a major global concern resulting from anthropogenic activities and natural geological processes. Their removal and recovery are important for protecting water resources and addressing the depletion of critical metals. This study evaluated wild sugarcane (WSC) biochar as an adsorbent for Cu2+ and Zn2+ removal from aqueous media, providing a potential valorization pathway for this biomass. WSC biochars were produced at airflow rates of 8, 12, 16, and 20 L min−1 and characterized by Fourier-transform infrared spectroscopy, Raman spectroscopy, and X-ray diffraction. Cu2+ and Zn2+ adsorption data were analyzed using the Langmuir, Freundlich, and Temkin isotherm models. The characterization results indicated the presence of oxygen-, carbon-, and hydrogen-containing surface functional groups that may contribute to metal adsorption. Under the investigated batch conditions, BP12 achieved removal efficiencies of approximately 70–90% for Cu2+ and 83–94% for Zn2+. For BP12, the Langmuir expression yielded an apparent maximum Cu2+ adsorption capacity of 42.3 mg g−1, while Zn2+ uptake reached 26.9 mg g−1 and was best described by the Freundlich expression among the models evaluated; the Langmuir model did not converge for Zn2+. These results demonstrate the potential of WSC biochar for Cu2+ and Zn2+ removal from aqueous media and support the valorization of wild-sugarcane biomass as a minimally processed adsorbent.

Applied Biosciences doi: 10.3390/applbiosci5040086

Authors: Surachai Techaoei Thisakorn Dumrongphuttidecha Wongnapa Nakyai Khemjira Jarmkom Warachate Khobjai

This study aimed to optimize pigment production from Monascus purpureus TISTR3090 using Khao Klong broken rice (brown rice) as a solid substrate and to evaluate the antioxidant and anti-aging activities of the resulting pigment extracts. Solid-state fermentation showed a progressive increase in red pigment production, reaching a maximum on day 8 (OD680 = 0.10). The water extract (WAE) exhibited a higher total phenolic content (148.86 mg GAE/g) than the ethanol extract (ETE) and demonstrated stronger antioxidant activities, including DPPH radical inhibition (74.66%) and ABTS scavenging activity (65.53%). In addition, WAE showed superior anti-aging potential, with greater inhibition of tyrosinase (75.29%) and collagenase (32.88%) compared to ETE. The MP extract exhibited the presence of R-NH2 functional groups, identified by FTIR, indicating the primary criterion for fungal red pigment. Gas chromatography–mass spectrometry (GC-MS) analysis identified 25 bioactive compounds, mainly fatty acid methyl esters and aromatic derivatives, which may contribute to the observed bioactivities. Liquid chromatography–mass spectrometry (LC-MS) further confirmed higher levels of gallic acid, chlorogenic acid, epigallocatechin gallate, and oroxylin A in WAE. Thus, Monascus pigments derived from broken brown rice-based fermentation represent a promising natural colorant and functional bioingredient with potential applications in nutraceutical and cosmeceutical industries, warranting further studies on formulation, stability, and in vivo validation.

Applied Biosciences doi: 10.3390/applbiosci5040085

Authors: Carla P. Morales-Guzmán Inés A. Cea-Pavez Inmaculada Moscoso-Ruiz David Manteca-Bautista Vito Verardo Martin Gotteland

Macroalgae are a valuable source of bioactive substances, particularly phenolic compounds with antioxidant properties. However, conventional extraction methods frequently rely on non-GRAS solvents, limiting their applicability in food and nutraceutical products. This study aimed to develop a Pyropia orbicularis extract enriched in bioactive compounds and antioxidant activity using GRAS solvents through a solid–liquid extraction process. Response surface methodology was employed to optimize the extraction process using total phenolic content (TPC) and FRAP assay as response variables. The selected maceration conditions were 80% ethanol, 55 °C, an algae-to-solvent ratio of 1:29.4 (g/mL), and 40 min of stirring. The resulting dried extract exhibited a TPC of 106.9 ± 3.5 mg GAE/g, a flavonoid content of 12.4 ± 0.3 mg QE/g, and antioxidant capacities of 50.0 ± 2.2 µmol TE/g by FRAP and 500.1 ± 8.9 µmol TE/g by ORAC. HPLC-MS analysis revealed a chemically diverse profile, with major compounds tentatively identified as amino acid derivatives, lipids, and organic acids. No cytotoxicity was observed in Caco-2 or HT-29 cells at concentrations up to 1000 µg/mL. Overall, the optimized extraction strategy enabled the recovery of a bioactive-rich extract from P. orbicularis, supporting its further investigation as a potential ingredient for food-related applications.

Applied Biosciences doi: 10.3390/applbiosci5040084

Authors: Stanislav Boychenko Anastasia Dakhnevich Tatiana Semerikova Alexander D. Egorov

Obesity is a global health threat, which continues to rise in prevalence worldwide, driving increases in related chronic diseases such as type 2 diabetes and cardiovascular conditions. While the sheer scale of the crisis affects over a billion people worldwide, the challenge is compounded for a distinct population: patients with genetically inherent obesity. Gene therapy targeting adipose tissue offers promise for treating obesity, but it requires development of tissue-targeting methods to minimize off-target effects and ensure safety. Our study presents two strategies to restrict transgene expression specifically to adipocytes: engineered adipose-specific promoters and optogenetic regulation. We designed truncated promoter fragments from murine Ucp1, Adipoq, and Fabp4 genes and evaluated their activity during 3T3-L1 preadipocyte differentiation. While all adipocyte-specific promoters exhibited substantially lower activity than the constitutive Cytomegalovirus promoter (10- to 100-fold reduction), they displayed distinct temporal activation patterns: Ucp1 peaked during active differentiation, whereas Adipoq was predominantly active in mature adipocytes. In vivo validation using AAV6/8 vectors confirmed adipose expression of the fluorescent reporter Katushka2S under the Ucp1 promoter in murine interscapular fat. Bioinformatic analysis (MAST/MCAST) of six adipocyte-enriched genes (Fabp4, Adipoq, Ucp1, Lep, Cidec, Sfrp5) identified high-density transcription factor binding site clusters that can be used for the development of new promoters for adipose-specific expression. Furthermore, we implemented the iLight optogenetic system to achieve light-dependent reassembly of split FOXP4 and PRDM16 transcription factors, demonstrating tunable Ucp1 activation with minimal basal expression. These strategies—development of synthetic adipose-specific mini-promoters and light-inducible regulation—may serve as a basis for developing precision metabolic medicine as these approaches allow spatiotemporal control of transgene expression.

Applied Biosciences doi: 10.3390/applbiosci5030083

Authors: Samira Rustamova Atabay Jahangirov Ulduza Gurbanova Faig Khudayev Jens Léon Ali Ahmad Naz Irada Huseynova

Drought during reproductive development and grain filling is a major constraint to bread wheat productivity in rainfed environments. In the present study, we employed genome-wide association analysis in an untapped diversity panel of wheat genotypes relevant to natural dryland conditions. A total of 187 genotypes were evaluated for drought-related physiological, biomass, and architectural traits under terminal rainfed stress in Azerbaijan. Relative water content, plant height, fresh weight, dry weight, flag leaf length, and flag leaf width were assessed at the milk ripening stage. These data were subjected to genome-wide association analysis using 19,737 SNP markers to identify loci and epistatic interactions involved in the determination of these traits. The panel showed broad phenotypic variation and significant genotypic effects for all traits, with broad-sense heritability ranging from 0.991 for plant height to 0.385 for flag leaf width. GWAS identified a major locus for relative water content on chromosome 2D at SNP marker AX-86184518, which explained 11.94% of the genotypic variation. Candidate-gene analysis identified the proximal WEB-family-like gene TraesCS2D03G1001000 as a putative positional candidate. Plant height showed strong additive loci, mainly on chromosomes 2A and 4A, whereas biomass and flag leaf traits showed suggestive additive loci and epistatic interactions. These findings identify candidate loci and putative interaction patterns associated with drought-related traits, which require further validation across independent populations and environments.

Applied Biosciences doi: 10.3390/applbiosci5030082

Authors: Camilo Zamora-Ledezma Dulexy Solano-Orrala Juan J. Leguineche Javier García-Molleja Juan P. Fernández-Blázquez Martin Bergaño-Guzmán Jesús Romero Pozuelo María Rosario Baquero Carmen Lorenzo-Aparicio José Manuel Martínez-Hernandez Daniela Negrete-Bolagay Victor H. Guerrero

This work evaluates agarose (AG)- and chitosan (CS)-based hydrogels incorporating a dexamethasone–cyclodextrin complex (Dex) as potential bioink candidates for wound healing applications. For this purpose, biopolymer hydrogels were formulated and tested to determine their structure, morphology, and rheological behavior under different strain, frequency, and temperature conditions, as well as their swelling behavior, surface functionality, protein adhesion capability, and antibacterial activity. Scanning electron microscopy revealed an interconnected membrane-like porous structure in the agarose/chitosan (BAC) hydrogels, which also showed a fibril-like microstructure when Dex was added. X-ray diffraction showed that these structures had a semicrystalline nature, with a main broad peak from approximately 15 to 20°. Rheological tests confirmed the viscoelastic behavior of the developed hydrogels. Chitosan incorporation reduced the rigidity of the formulations, whereas dexamethasone did not significantly affect the viscoelastic response of the BAC hydrogel. Additionally, the temperature sweep showed that Dex did not substantially alter the viscoelastic response of the BAC hydrogel while reducing mass loss after immersion in phosphate-buffered saline and not significantly affecting the swelling behavior. Further, Coomassie blue staining tests demonstrated favorable protein interaction with the BAC-based hydrogels, indicating that Dex incorporation did not compromise their adsorption capacity. Regarding their antibacterial activity, the hydrogels showed no major effects on Escherichia coli and Staphylococcus aureus in disk diffusion tests. However, liquid diffusion tests showed almost no bacterial growth for hydrogels incorporating CS. Thus, the present results demonstrate the potential of the BAC hydrogel system for biomedical and cosmetic applications.

Applied Biosciences doi: 10.3390/applbiosci5030081

Authors: Sabia Khan Md. Abdullah Al Sabbir Nabela Akter Ankita Saha Imran Khan Yuan Xu Mohammad Golam Mostofa Md. Motaher Hossain

Extreme temperatures, drought, and salinity are among the most detrimental abiotic stressors limiting global plant productivity, and their frequency has intensified under climate change. These escalating pressures underscore the need for sustainable biological strategies that enhance plant resilience to climate-induced abiotic stresses. Plant growth-promoting rhizobacteria (PGPR) have emerged as a promising, eco-friendly solution due to their ability to optimize rhizospheric processes that strengthen plant adaptive capacity. PGPR improve nutrient acquisition, maintain ionic homeostasis, modulate phytohormone signaling, and regulate ethylene levels through ACC deaminase activity. They also stimulate antioxidant defenses, promote osmolyte and exopolysaccharide synthesis, and enhance root system development—key traits that collectively alleviate drought, salinity, and heat stress. Recent research demonstrates that co-inoculation, multi-strain microbial consortia, and synthetic communities designed using multi-omics approaches significantly enhance PGPR stability, colonization, and functional effectiveness under field conditions. Additionally, nanotechnology-enabled formulations and smart delivery systems are emerging as innovative tools to improve PGPR survival and targeted release in harsh environments. This review synthesizes current insights into PGPR-mediated stress mitigation, highlights technological innovations that support their application, and outlines pathways for integrating PGPR into climate-resilient, sustainable agricultural systems to safeguard crop productivity amid escalating environmental stress.

Applied Biosciences doi: 10.3390/applbiosci5030080

Authors: Liliya Stoyanova Margarita Docheva Desislava Kirkova Maria Angelova-Romova

Tobacco seeds (Nicotiana tabacum L.) represent a promising nicotine-free source of bioactive compounds, particularly polyphenols and natural antioxidants. This study evaluated the total phenolic content (TPC) and antioxidant activity of tobacco seeds, defatted seed cake, and seed oil obtained from oriental tobacco varieties cultivated under organic and conventional conditions over two consecutive years (2020–2021). Extracts from tobacco seed-derived products were prepared using different solvents (water, 60% methanol, 80% methanol, 80% ethanol, and 95% ethanol). TPC was determined by the Folin–Ciocalteu method, while antioxidant capacity was assessed by DPPH, ABTS, and FRAP assays. In addition, the Relative Antioxidant Capacity Index (RACI) and the Phenolic Antioxidant Coefficient (PAC) were applied to compare the antioxidant performance of the different samples and to estimate the contribution of phenolic compounds to the overall antioxidant activity under the two cultivation conditions. The results showed that extraction efficiency strongly depended on solvent polarity, with 60% methanol providing the highest recovery of phenolic compounds from seeds and seed cake, while 80% ethanol was more effective for oil. Seed cake extracts exhibited higher TPC (1.15 ± 0.04 mg GAE g−1–3.98 ± 0.10 mg GAE g−1) and antioxidant activity than seed extracts (0.070 ± 0.04 mg GAE g−1–2.02 ± 0.02 mg GAE g−1) and oil (0.29 ± 0.01 mg GAE g−1–1.70 ± 0.17 mg GAE g−1), indicating enrichment of phenolic compounds after oil removal. Among the tested extraction conditions, 95% ethanol and 60% methanol extracts showed the strongest antioxidant potential, with maximum FRAP (23.10 mM TE g−1), DPPH (3.35 mM TE g−1), and ABTS (14.70 mM TE g−1) values depending also on variety and harvest year. Organically produced samples showed higher TPC and RACI values than conventionally produced materials, while PAC analysis revealed the strongest association between phenolic compounds and antioxidant capacity in the FRAP assay. These findings provide a comparative evaluation of the antioxidant potential of tobacco seed-derived fractions and the effectiveness of the extraction methods used for their recovery.

Applied Biosciences doi: 10.3390/applbiosci5030079

Authors: Anton Bahtiar Sekar Ayu Rabbani Asfareni Rahmah Sirithon Siriamornpun

Obesity is a multifactorial metabolic disorder characterized by dysregulated energy homeostasis, impaired lipid metabolism, and altered hypothalamic appetite signaling. This study aimed to evaluate the anti-obesity effects and underlying mechanisms of a 95% ethanol extract of Artocarpus integer stem bark in a high-fat diet (HFD)-induced obese rat model. Obesity was induced in male Wistar rats using HFD feeding, followed by treatment with the extract (200–800 mg/kg/day, oral). Semaglutide and resveratrol were used as reference compounds. Body weight, food intake, adiposity, serum lipid profile, adipokines, histological changes, and gene expression related to lipid metabolism (Fas, Pparγ, Cpt-1, AdipoR1) and hypothalamic appetite regulation (Pomc, Mc4r, Npy) were evaluated. Phytochemical profiling was performed using LC–HRMS. The extract markedly reduced body weight gain, visceral adiposity, and food intake compared with the HFD control (p < 0.05), with the high-dose extract demonstrating a substantial weight-reducing effect that approached the magnitude of semaglutide. Treatment improved dyslipidemia by decreasing total cholesterol and triglycerides while increasing HDL levels. Adipokine imbalance was restored, as evidenced by reduced leptin and amylin and increased adiponectin levels. At the molecular level, the extract downregulated lipogenic genes (Fas, Pparγ), partially restored Cpt-1 expression, and normalized AdipoR1. In the hypothalamus, Pomc and Mc4r were upregulated, whereas Npy was suppressed. LC–HRMS analysis revealed the presence of stilbene, chalcone, and flavonoid derivatives. In conclusion, A. integer stem bark extract exerts anti-obesity effects through coordinated modulation of appetite signaling and lipid metabolism, supporting its potential as a multi-target phytotherapeutic agent for obesity management.

Applied Biosciences doi: 10.3390/applbiosci5030078

Authors: Ayoub Lafnoune Saad Harrizi Jihane Ait Benbella Asmaa Chbel Hicham Wahnou Ismail Guenaou Bouchra Darkaoui Imane Nait Irahal

Tumor angiogenesis plays a critical role in cancer growth and metastatic progression and therefore represents an important therapeutic target. Bee venom-derived peptides constitute a diverse source of bioactive molecules whose interactions with angiogenesis-associated receptors remain incompletely characterized. In this study, an integrated in silico workflow was applied to investigate the potential interactions of selected bee venom peptides, including melittin and tertiapin, with major angiogenic-associated receptors, including platelet-derived growth factor receptor alpha (PDGFR-α), vascular endothelial growth factor receptors, fibroblast growth factor receptors, and integrin αvβ3. Molecular docking was used to identify and prioritize potential peptide–receptor complexes, while molecular dynamics simulations were performed to assess their conformational stability and interaction patterns under the simulated conditions. Reactome pathway enrichment analysis was subsequently used to contextualize the selected molecular targets within angiogenesis-related biological processes. Melittin–PDGFR-α and tertiapin–integrin αvβ3 emerged as the highest-ranked complexes and exhibited relatively stable interaction patterns with persistent intermolecular contacts throughout the simulations. Pathway enrichment analysis further associated the investigated targets with PDGFR signaling, cell migration, and other angiogenesis-related processes. Collectively, these findings suggest that melittin–PDGFR-α and tertiapin–integrin αvβ3 represent promising peptide–receptor pairs for further investigation while providing a computational rational for exploring bee venom peptides as potential modulators of tumor angiogenesis. However, these findings should be considered hypothesis-generating and primarily serve to prioritize peptide-receptor pairs for future experimental validation, including binding assays, cellular assays, and relevant in vivo studies. Peptide selectivity, toxicity, stability, and targeted delivery should also be evaluated before any therapeutic application can be considered.

Applied Biosciences doi: 10.3390/applbiosci5030077

Authors: Wattana Weerachatyanukul Orawan Thongsum Pitchanee Jariyapong

Virus-like particles (VLPs) are nanoscale platforms that can be engineered for heterologous molecule display. Among the available VLP platforms, Macrobrachium rosenbergii nodavirus-derived VLPs (MrNV VLPs) have emerged as a promising system for this purpose. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, 20 eligible studies were identified and qualitatively synthesized to summarize the current design strategies used for heterologous molecule display on MrNV VLPs, with a particular focus on how insert characteristics influence the selection of design strategies. The protruding (P) domain, located at the C-terminal region, serves as the main site for heterologous display. Different strategies, including genetic fusion, peptide insertion, and domain replacement, are applied depending on the size and structural complexity of the insert. Across the available studies, these approaches enabled the display of both small peptides and larger protein domains, with particle architecture maintained in studies that directly evaluated and reported VLP formation. In addition to design strategies, physicochemical properties, including structural stability, are important considerations that may influence platform performance. However, several studies did not directly report VLP assembly, and the available evidence remains heterogeneous and predominantly preclinical. Together, this review highlights the relationship between insert characteristics and design strategies in MrNV VLPs and provides insights for the rational design of VLP-based heterologous molecule display platforms for biomedical applications.

Applied Biosciences doi: 10.3390/applbiosci5030076

Authors: Sasikan Channgam Gad Elsayed Mohamed Salem Thatsanee Luangharn Supachai Pisuchpen Thomas Karbowiak Pornchai Rachtanapun Wirongrong Tongdeesoontorn

Increasingly, mycelium-based biomaterials are being investigated as sustainable alternatives to conventional packaging materials, but their inherent brittleness limits their practical application. The present study investigated the properties of pure mycelium sheets sourced from Ganoderma sp., Lentinus sp., and Pleurotus sp., employing glycerol plasticization and zinc oxide nanoparticles (ZnONPs). The findings revealed that growth and structural attributes varied according to the species, with Ganoderma and Lentinus exhibiting enhanced colonization rates. Scanning electron microscope (SEM) examination indicated that Lentinus developed dense fiber networks, Ganoderma exhibited compact structures, while Pleurotus presented a porous morphology. Glycerol enhanced flexibility but diminished tensile strength, while ZnONPs offered marginal reinforcement. Lentinus exhibited the greatest density, whereas untreated Ganoderma exhibited the highest thermal stability among the control samples (284.97 °C), while glycerol-treated Pleurotus exhibited the highest thermal stability overall (287.39 °C). Glycerol-treated sheets demonstrated improved ductility and heightened hydrophilicity. The properties of mycelium are species-dependent and can be modulated through targeted post-treatment strategies. Lentinus and Ganoderma exhibit significant potential as sustainable bio-based materials for packaging applications. These findings demonstrated that Lentinus sp. treated with glycerol achieved the optimal balance of flexibility and density for sustainable packaging, while glycerol-treated Pleurotus sp. exhibited superior thermal stability.

Applied Biosciences doi: 10.3390/applbiosci5030075

Authors: Petya Georgieva Petya Peneva Yana Gvozdeva

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the management of inflammatory disorders, pain, and fever due to their significant anti-inflammatory and antipyretic properties. However, their long-term use is often associated with multiple adverse effects, including gastric erosion, hemorrhage, and perforation, as well as cardiovascular, hepatic, and renal complications. Indomethacin is a potent NSAID commonly used in the treatment of pain and rheumatoid arthritis. Its poor water solubility, Biopharmaceutics Classification System (BCS) Class II, results in low aqueous solubility and high membrane permeability, and conventional dosage forms often fail to overcome pharmacokinetic limitations and gastrointestinal irritation. To address these drawbacks, alternative indomethacin-loaded drug delivery systems have been developed, which enable controlled release, enhanced solubility, and targeted delivery for improving therapeutic efficacy and safety profile. Nature provides a diverse reservoir of bioactive compounds with significant anti-inflammatory potential, making them promising alternatives or complementary agents to indomethacin. Representative examples include the alkaloids piperine and berberine; the flavonoids oleocanthal and apigenin; and the terpenoids thymoquinone, kahweol, and cafestol; as well as anthraquinones and iridoids. This review summarizes the current evidence on plant-derived anti-inflammatory compounds that may “replace” or act synergistically with indomethacin. In addition, we discuss the pharmacological properties of indomethacin and recent advances in drug delivery systems designed to improve its topical and systemic therapeutic applications.

Applied Biosciences doi: 10.3390/applbiosci5030074

Authors: Omorovbiye C. Odeh Emmar E. Okpakpor Samuel Frank Israel O. Bolanle Raymond I. Ozolua

Psychotic disorder remains a major global health burden despite the plethora of anti-psychotic medications currently available. Poor adherence owing to numerous adverse effects and cost burden is a major challenge to treating psychosis. In view of this, the use of ethnomedicinal plants has become more common. In Nigeria, West Africa, the seeds of Phaseolus lunatus (Lima bean) are used to treat psychosis-like illness. Despite its extensive use, there are not many scientific studies that validate its efficacy. Therefore, this study evaluated the antipsychotic, anxiolytic-like, and sleep-enhancing properties of the seed extract. Pulverized dried seeds were extracted in a mixture (1:1) of water and methanol and screened for phytoconstituents. Using murine models, oral doses of 400, 800, and 1600 mg/kg of the extract were evaluated for antipsychotic, anxiolytic, and sleep-enhancing properties. Brain malondialdehyde (MDA), superoxide dismutase (SOD), catalase, glutathione peroxidase, and glutathione reductase (GR) levels were estimated after administering the doses of extract to mice for 14 consecutive days. The seed extract significantly inhibited d-amphetamine-induced stereotypy and ketamine-induced hyperlocomotion in a dose-dependent manner. It also exhibited significant anxiolytic-like activity at 800 mg/kg, as indicated by increased time spent on the open arm of the elevated plus maze and an increased number of head dips on the hole board. Compared with untreated controls, sleep onset was significantly faster, and sleep duration was longer in mice that received 1600 mg/kg of the extract. There was a decrease in MDA levels at a dose of 1600 mg/kg and an increase in SOD and GR levels in the mice brains. Our findings suggest that Lima bean extract exhibited antipsychotic-like, anxiolytic-like, sedative–hypnotic, and antioxidant activities in mice likely due to its flavonoid and alkaloidal constituents. Our study provides preliminary evidence in mice that supports the ethnomedicinal use of Lima beans as a functional food for psychosis-like illness.

Applied Biosciences doi: 10.3390/applbiosci5030073

Authors: Rosário Costa Ana C. Silva Ana Portela João C. Ferreira Patrícia T. Pires Paulo Melo

Background: This systematic review aimed to evaluate the effect of dentin pretreatment strategies on bond strength to natural caries-affected dentin (NCAD) in permanent human teeth. Methods: A systematic search was conducted in PubMed, Web of Science, and Scopus databases up to April 2026, following PRISMA guidelines. Experimental and quasi-experimental in vitro studies evaluating the bond strength of resin restorations to NCAD after the application of dentin pretreatment strategies prior to adhesive procedures were included. Studies involving modified adhesive systems, artificial caries-affected dentin, or primary teeth were excluded. Risk of bias was assessed using the QUIN tool. Due to methodological heterogeneity, a qualitative synthesis was performed. Results: A total of 1071 studies were identified, of which 9 met the inclusion criteria. Pretreatment strategies were categorized into cross-linking/biomodulation, remineralization, and fluoride-based approaches. Cross-linking strategies, particularly flavonoid-based agents, consistently improved bond strength. Remineralization approaches, including CPP-ACP, demonstrated condition-dependent effects, particularly under aging conditions. Fluoride-based strategies showed more variable outcomes. Most included studies presented moderate risk of bias. Conclusions: Cross-linking/biomodulation strategies demonstrated the most favourable bond strength outcomes to NCAD, while remineralization approaches showed potential benefits under aging conditions. However, the available evidence remains limited and methodologically heterogeneous, indicating the need for further standardized studies.

Applied Biosciences doi: 10.3390/applbiosci5030072

Authors: Irene Sinagridi Eleni Andreou

Skin aging is a multifactorial process involving structural and functional changes in the dermis. In recent years, the treatment of age-related skin changes in the face, neck, and décolleté has become a growing focus in the fields of esthetic medicine and dermatology. The increasing demand for natural, youthful results without surgical intervention or prolonged downtime has led to the development and widespread application of minimally invasive energy-based technologies, such as High-Intensity Focused Ultrasound (HIFU) and Radiofrequency (RF). The aim of this review is to summarize current evidence regarding the mechanisms of action, clinical efficacy, and safety of HIFU and RF, as well as their combined applications in skin rejuvenation. Relevant studies were selected based on their scientific quality and relevance to the topic. Current evidence indicates that both HIFU and RF promote collagen remodeling and dermal tightening through different energy-delivery mechanisms. Comparative studies suggest that their combined use may enhance esthetic outcomes and improve patient satisfaction. Overall, these technologies represent innovative, non-surgical approaches for facial, neck, and décolleté rejuvenation.

Applied Biosciences doi: 10.3390/applbiosci5030071

Authors: Monika Szczecińska Aniela Garbowska Natan Pupek Wiktoria Czochór Kamil Koczwara Joanna Szablińska Jakub Sawicki

Reliable molecular identification of plants depends on reference libraries of DNA sequences, and complete plastid genomes (plastomes) used as super-barcodes are increasingly the reference of choice for taxonomically difficult groups. We assess how ready the flora of Poland is for plastome-based super-barcoding by combining national checklists of vascular plants, mosses, and liverworts with the current availability of complete plastomes in GenBank. For 3339 species we quantify coverage, its distribution across families and genera, the provenance of the sequenced material, and the gap between coverage and expected species-level resolution. A complete plastome was available for 54.4% of species, but coverage was uneven: liverworts (76.0%) and mosses (75.6%) were close to reference-complete, whereas the much larger vascular flora reached only 45.9%. Effective vascular coverage fell to roughly 39% once low-resolution genera were excluded, because many uncovered species belong to apomictic or hybridogenous complexes such as Taraxacum, Hieracium, and Alchemilla, where plastomes alone cannot resolve identity. Most sequenced material was of non-European origin, which weakens fine-scale identification of local populations. We identify 1011 high-value sequencing targets and recommend a tiered, marker-aware strategy combining the plastome with nuclear ribosomal DNA and, selectively, low-copy nuclear or mitochondrial markers. Defining readiness by the proportion of the flora that can actually be identified turns a static inventory into a prioritized roadmap transferable to other Central European floras.

Applied Biosciences doi: 10.3390/applbiosci5030070

Authors: Maria Teresa Colangelo Marco Meleti Stefano Guizzardi Carlo Galli

Scaffold architecture shapes tissue regeneration through the mechanical, topographical, and biochemical cues it presents to cells, yet geometrically elaborate scaffolds do not reliably produce more organized tissues, while comparatively simple architectures can exert strong organizational effects. We argue that scaffold performance is better understood by distinguishing geometric complexity from effective informational dimensionality: a relational property of the scaffold–cell system, defined as the number of independently manipulated architectural directions that produce distinguishable, above-noise changes in a jointly measured mechanotransductive response. Unlike structural entropy, fractal dimension, or feature-counting metrics, this construct depends on cellular accessibility, cue persistence, and non-redundancy. Mechanotransduction supplies its biological basis, integrating scaffold-derived cues through focal adhesions, cytoskeletal organization, nuclear deformation, and YAP/TAZ signaling, and we distinguish early resolvability from later organizational stabilization. We outline an operational strategy for estimating both from factorial scaffold libraries, common readout panels, and rank-based analysis of the response mapping, illustrated with selected experimental precedents rather than a systematic evidence sample. Positioned relative to biomimetic, mechanobiology-guided, and morphospace approaches, it yields testable predictions on dimensional compression, redundancy, and the resolvability–stability dissociation. Scaffold design is thus reframed from maximizing complexity or native resemblance toward engineering stable, cell-readable dimensions of organization.

Applied Biosciences doi: 10.3390/applbiosci5030069

Authors: Wushan T. Jayalath Medha J. Gunaratna Chamila C. Kadigamuwa Charitha Thambiliyagodage Jayangika N. Dahanayake

Globally, over 55 million people are affected by Alzheimer’s disease (AD), a number that is increasing rapidly and projected to triple by 2050. AD is a type of neurodegenerative disorder for which there is no curative treatment. The accumulation of amyloid-β (Aβ) plaques and the formation of neurofibrillary tangles are the hallmarks of AD. Curcumin, a bioactive compound extracted from turmeric rhizomes, has shown significant neuroprotective properties, including anti-amyloidogenic, anti-inflammatory, antioxidant, and metal-chelating. Though much research has revealed the potential of curcumin as a promising therapeutic agent for AD, the limited bioavailability of curcumin restricts its effectiveness in pharmaceutical applications. To overcome this challenge, nanocarrier-based delivery systems have emerged as a promising strategy for enhancing the bioavailability and therapeutic efficacy of curcumin. This review provides an overview of recent findings on curcumin-based nanocarriers for AD therapy.

Applied Biosciences doi: 10.3390/applbiosci5030068

Authors: Tien Cuong Nguyen Thuy Hang Dam Thi Thao Hoang Thi Hong Van Nguyen Hai Van Nguyen Phu Ha Ho Hong Nga Luong

This study explored the microbial diversity of Vietnamese cocoa fermentation and the antifungal potential of indigenous bacteria. Cocoa beans were collected from three Vietnamese regions at different fermentation stages. Bacteria were isolated and identified using 16S rRNA gene sequencing. A total of 22 lactic acid bacteria (LAB) and 9 acetic acid bacteria (AAB) strains were identified, belonging to the genera Lactiplantibacillus, Limosilactobacillus, Levilactobacillus, Lacticaseibacillus, Pediococcus, Acetobacter, Gluconobacter, and Komagataeibacter. Four strains, including three LAB strains (L. plantarum L11E48, L. plantarum L15E48, L. brevis L8DN24) and one AAB strain (A. ghanensis G63E48), were evaluated for their ability to inhibit Aspergillus flavus and Fusarium foetens on agar media and bread. All strains demonstrated strong antifungal activity, inhibiting 51–100% mycelial growth of both fungi after 7 days of incubation. The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of cell-free supernatant were 640 μL/mL against A. flavus and varied from 160 to 320 μL/mL against F. foetens. When applied in breadmaking, L. plantarum L15E48, L. brevis L8DN24, and A. ghanensis G63E48 delayed fungal growth for 1–2 days. These findings provide valuable baseline data on Vietnamese cocoa fermentation microbiota and highlight the potential of indigenous LAB and AAB strains for biocontrol, food preservation applications, and starter culture development.

Applied Biosciences doi: 10.3390/applbiosci5030067

Authors: Blanca Aurora Francisco-Ponce Yanik Ixchel Maldonado-Astudillo Iris Paola Guzmán-Guzmán Gerardo Huerta-Beristain Gerónimo Arámbula-Villa Verónica Flores-Casamayor José Juan Véles-Medina Patricia Alvarez-Fitz Mónica Ramírez Enrique Flores-Andrade Ricardo Salazar Javier Jiménez-Hernández

Ultrasound technology has emerged as a promising non-thermal approach for modifying the structural and functional properties of food matrices. However, its impact on starch digestibility remains insufficiently understood, particularly in complex systems such as cereal, legume, and pseudocereal flours. This study evaluated the effect of power ultrasound on the functional properties and in vitro digestibility of durum wheat (DWF), chickpea (CF), and amaranth (AF) flours. Flours were treated using an ultrasonic probe (20 kHz, 60% amplitude) for 10 and 20 min while maintaining the sample temperature at 15 °C. Structural and functional properties were assessed, including color, morphology, water absorption, FT-IR spectra, pasting behavior, thermal properties, and starch fractions. Ultrasound induced structural modifications, including starch granule disruption and increased surface roughness. Lightness increased in DWF, CF, and AF, although the magnitude of the response differed among flour types. Water absorption improved in DWF but decreased in CF and AF. FT-IR spectra suggested molecular rearrangements, while viscoelastic and thermal analyses showed increased viscosity in DWF and CF and reduced viscosity in AF. Ultrasound also modified starch fractions and in vitro starch digestibility in a flour-dependent manner, reflecting the distinct structural responses of cereal, legume, and pseudocereal starches to acoustic cavitation. Overall, ultrasound modified the functionality of flour and starch digestibility in a matrix-dependent manner, supporting its potential as a clean-label technology for tailoring the functional properties of different flour matrices.

Applied Biosciences doi: 10.3390/applbiosci5030066

Authors: Maha Ezzelarab Midhila Madhusoodanan Shrimanti Ghosh Geetika Vadali Jacob L. Jaremko Abhilash Hareendranathan

Machine learning using supervised approaches has been widely applied to ultrasound image analysis. In contrast, reinforcement learning (RL), which is well suited for sequential decision-making, is underexplored. Ultrasound workflows involve sequential subtasks like image acquisition, quality assessment, summarization, and interpretation that can be integrated into RL frameworks. This scoping review examined RL applications in ultrasound. A comprehensive search was conducted in Scopus, PubMed, Embase, and MEDLINE for studies published between 2015 and 2026. Eligible studies used RL with clinical ultrasound data, and the data were summarized by application area, methods, and anatomical targets. The review also provides a brief overview of RL concepts as foundational knowledge for understanding various RL formulations used. From 326 records retrieved, 39 studies were included. Most studies used model-free deep RL, with value-based methods being the most common, particularly Deep Q-Network (DQN) and its variants, on retrospective data across diverse anatomical targets, with breast, fetal, and uterine ultrasound being the most frequently represented categories. RL was used to automate tasks including navigation, plane localization, landmark detection, and video summarization. RL in ultrasound imaging is an emerging field of research and has advantages for sequential workflow optimization tasks. Most approaches are at an early stage and have been tested on small datasets, lack consistent evaluation protocols, and report limited clinical translation.

Applied Biosciences doi: 10.3390/applbiosci5030065

Authors: Winy Cuchuyrumi-Nina Eliana Molina Mamani María Isabel Cajo-Pinche Javier Diaz-Viteri Ana María Muñoz Fernando Ramos-Escudero

Sinami (Oenocarpus mapora H. Karst) is a palm tree that grows in the South American Amazon, whose fruits are highly valued for their sensory characteristics, attractive dark purple color, and as a source of edible oil. However, little to no information is available on the changes in the physicochemical properties, nutrients, chemical compounds, and antioxidant capacity of O. mapora fruits throughout their four-week ripening period. The results obtained showed that fruit weight, seed weight, and peel + flesh weight increased slightly. The variables that showed a significant increase (p < 0.001) during ripening were pH, TSS (°Brix), TSS/TA ratio, and the a* coordinate. The lipid content increased substantially from 0.46 to 31.47 g/100 g. Among the variations, the chlorophyll and carotenoid content increased from harvest 1 to harvest 2, while the total anthocyanin content decreased from harvest 3 (1.65 mg c-3-g/g) to harvest 4 (1.00 mg c-3-g/g). Furthermore, 4-hydroxyphenylacetic acid and ferulic acid levels decreased, while gallic acid and rutin levels increased. These results confirm that the fruits of O. mapora vary during the ripening process. Harvests 3 and 4 contributed significantly with a higher content of bioactive compounds and oil, which is important for the utilization and production of vegetable oil and for promoting the use of byproducts such as seeds and the press cake after oil extraction.

Applied Biosciences doi: 10.3390/applbiosci5030064

Authors: Lorraine Sallah Patrick W. Narkwa Seth A. Domfeh Peter N. Coffie Patience N. Ansong Cynthia A. Danquah Kofi O. Owusu-Daaku Babatunde M. Duduyemi

Mentha pulegium L. is reported to contain phytochemicals known to bind to estrogen receptors and modulate estrogenic effects. A hydroethanolic leaf extract of Mentha pulegium L. (MPE) was prepared, and its effects on uterine and ovarian tissues in immature rats were investigated, focusing on transcriptional endpoint-related estrogenic activity. Female Sprague Dawley rats were treated with varying doses of MPE alone or in combination with estradiol for seven days. Gene expression analysis was performed using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) to evaluate the effect of MPE on estrogen-responsive biomarkers: Calbindin-D9k (CaBP-9k), Progesterone receptor (Pgr), Trefoil factor 1 (pS2), Intestinal calcium-binding protein integral (Icabp), Integral membrane-associated protein-1 (Itmap1) and Complement component 3 (CC3) genes. In ovarian tissues, MPE treatment decreased CaBP-9k and Icapb expression, with CC3 showing significant decreases in the 200 mg/kg group. Treatments with MPE and estradiol significantly reduced the expression of all estrogen-responsive genes compared to estradiol treatment. In uterine tissues, 1000 mg/kg MPE increased CaBP-9k and pS2 expression significantly but decreased Icapb, CC3, pS2, and Itmap across all treatment groups significantly. Combined estradiol treatment with MPE (500 and 1000) mg/kg showed significantly low CaBP-9k and CC3 expressions. Increased expression of Icapb, Itmap, and pS2 was observed when combined estradiol treatments with MPE (500 and 1000) mg/kg were compared to estradiol treatment. MPE influenced the expression of specific genes in the uterus and ovaries and thus may exhibit endocrine-modulatory activity by multiple mechanisms of action, highlighting its potential complexity in modulating estrogenic responses.

Applied Biosciences doi: 10.3390/applbiosci5030063

Authors: Rubén Gil-Gonzalo Palmira Villa-Valverde Inmaculada Aranaz David Castejón

In the valorization of organic waste within circular bioeconomy frameworks, compost and insect frass are increasingly used as soil amendments, yet their molecular composition and the metabolic signatures of biological processing remain poorly characterized. In this study, high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) was applied to intact compost and frass samples to explore their metabolic profiles and assess the potential of this technique to detect bioindicators of vermi- and insect-associated biotransformation without destructive sample preparation. HRMAS NMR experiments included water-suppressed one-dimensional 1H spectra and selected two-dimensional experiments (1H–13C HSQC and 1H–13C HMBC) to support signal assignment. The analysis revealed clear differences between compost with and without vermicomposting activity, with low-molecular-weight resonances assigned to glycine betaine (betaine), a characteristic metabolite associated with biotransformation. In Tenebrio molitor frass, 1H-HRMAS spectra displayed a distinct set of small-molecule signals, including betaine resonances detected for the first time in this matrix. Time-resolved monitoring showed that betaine signals remained stable during the studied period. These findings demonstrate that HRMAS NMR provides a minimally invasive, versatile platform for direct characterization of compost and frass, supporting the identification of metabolic signatures linked to vermi- and insect-mediated processing.

Applied Biosciences doi: 10.3390/applbiosci5030062

Authors: Ivana Šutej Krešimir Bašić Matej Kurkutović Kaltrina Smajli Vokshi Jasmina Šipkar Karla Sučević Kristina Peroš

Salivary calcium is a biologically plausible but methodologically challenging biomarker because of its central role in enamel demineralization/remineralization, dental calculus formation, and oral mineral homeostasis. This narrative review summarizes the analytical challenges and clinical interpretation of salivary calcium measurements in oral and systemic disease. Evidence from periodontal research generally indicates higher salivary calcium concentrations in periodontitis, although interpretation is complicated by calculus formation, gingival crevicular fluid contribution, smoking, inflammation, and variation in saliva collection protocols. In dental caries, findings are heterogeneous, and calcium appears more meaningful when interpreted together with salivary flow rate, pH, phosphate, buffering capacity, and fluoride exposure than as an isolated diagnostic marker. Studies in osteoporosis, chronic kidney disease, hemodialysis, and diabetes suggest possible associations with systemic disturbances, but salivary calcium does not currently provide sufficient specificity or predictive value to replace established serum, imaging, or clinical biomarkers. Overall, salivary calcium should be regarded as a context-dependent component of broader salivary biomarker panels. Its clinical translation requires standardized sampling, clear distinction between total and ionized calcium, appropriate analytical validation, and careful control of pre-analytical and biological confounders.

Applied Biosciences doi: 10.3390/applbiosci5030061

Authors: Elizabeth Refugio-García Zaira Itzel Bedolla-Valdez Alfredo Emiliano Chávez-Pantiga Gerardo Vázquez-Huerta José Guadalupe Miranda-Hernández Carlos Adrián Calles-Arriaga José Amparo Rodríguez-García Enrique Rocha-Rangel

Hydroxyapatite (HA) is widely used in biomedical applications due to its biocompatibility and chemical similarity to the mineral phase of bone; however, its low mechanical strength limits its structural use. In this work, HA ceramics with different Mg additions (0, 0.5, 1, 3, 5, and 10% by weight) were prepared using the powder processing technique. The mixtures were homogenized, conformed and sintered at 1100 °C. The incorporation of intermediate Mg concentrations produced an increase in fracture toughness compared to pure HA. The best mechanical performance was obtained with the formulation containing 5% Mg by weight, achieving a hardness of 319 HV, a porosity of 12.92% and a fracture toughness of 4.06 MPa·m0.5, comparable to those reported for human cortical bone, indicating its potential for applications in moderately loaded bone implants. The findings indicate that magnesium functions as a reinforcing component in the ceramic matrix, mitigating critical defects and thereby contributing to the improved toughness of Mg-containing hydroxyapatite ceramics. The polarization resistance results show that the incorporation of low fractions by weight of magnesium (1% Mg) adjusts the electrochemical behavior of the material, while higher increases in its concentration cause a deterioration of this property.

Applied Biosciences doi: 10.3390/applbiosci5030060

Authors: Majd M. Alsaleh Dana A. Alqudah Bassam I. El-Eswed Mamoon M. D. Al-Rshaidat

Biofilm formation of ESKAPE pathogens creates a major problem in healthcare-associated infections, and nutrient supply significantly contributes to biofilm structure and bacterial survival. An understanding of how various nutritional environments influence polymicrobial biofilm dynamics is important for the development of targeted antimicrobials. In the present work, biofilm formation of ESKAPE pathogens was tested under three different nutritional environments—nutrient broth with no supplementation (NB; minimal), nutrient broth with supplementation of 2% glucose + 2% sucrose (NB+G+S; moderate), and Brain Heart Infusion supplemented with 2% glucose + 2% sucrose (BHI-G+S; Rich). Furthermore, we studied the effect of bacterial mixtures on biofilm features in each medium. Standard ATCC strains of Staphylococcus aureus (S), Enterococcus faecalis (E), Klebsiella pneumoniae (K), and Pseudomonas aeruginosa (P) were grown alone or in combination as mono-, dual-, tri-, and tetra-species. Biofilm formation was determined by crystal violet spectrophotometric assay (n = nine biological replicates, with three technical replicates each). LIVE/DEAD staining and confocal laser scanning microscopy (CLSM) were used for samples with absorbance values > 0.5, including 3-D architectural analysis of viability using ImageJ software. Biofilm height was quantified by using the ZEN depth code tool. Statistical analysis included one-way ANOVA with Tukey’s post hoc test (p < 0.05), and unpaired t-tests were performed when applicable to compare variables. Biofilm formation was drastically increased in rich medium compared to moderate and minimal NB. The tetra-species cocktail SKEP reached the highest biofilm dry weight in BHI+G+S and greater elevation/viability compared to NB+G+S, whereas P. aeruginosa monospecies exhibited an opposite trend (greater elevation in NB+G+S than BHI+G+S). Interestingly, the E. faecalis and P. aeruginosa group exhibited strong biofilm characteristics in a range of nutritional environments. Confocal analysis showed structurally distinct biofilms: dense, vertical biofilms in BHI+G+S and sparse, horizontal patterns in NB+G+S. Nutrient composition significantly impacts polymicrobial biofilm formation, with nutrient-rich conditions (BHI+G+S) supporting robust viable three-dimensional structures, particularly within complex bacterial communities. These observations are of potential significance with respect to biofilm formation in hospital settings, such as in wounds, and may be used in strategies against the targeted intervention of polymicrobial infections.

Applied Biosciences doi: 10.3390/applbiosci5030059

Authors: Sara Botezan Răzvan Ștefan Lucian Barbu-Tudoran Septimiu Cassian Tripon Marin Șenilă Victorița Bonta Daniel Severus Dezmirean

Royal jelly (RJ) is a nutrient-rich, bioactive substance that has been used for medicinal purposes since antiquity. For honeybees, RJ plays a nutritional role and provides structural support for queen larvae in their upside-down oriented hive cells. In recent decades, composite materials have become an increasingly important topic in materials science and tissue engineering. The present interdisciplinary study explores the development and characterization of composite materials consisting of strontium (Sr)-doped phosphate glass powders and RJ, proposing a novel approach that integrates the biocompatibility of phosphate glass materials with the bioactive properties of RJ. Three RJ samples were biochemically characterized, and the native ultrastructure of RJ was highlighted through transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The glass samples were characterized through dissolution tests, pH measurements, scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDX) and inductively coupled plasma optical emission spectroscopy (ICP-OES). The ultrastructure of RJ–glass mixtures was analyzed with TEM. This preliminary structural analysis allowed us to gain insight into the interactions between the two components. The landscape of the native RJ ultrastructure is dominated by proteinaceous fibril bundles embedded in a fibrillary ground substance. Our morphological observations indicate that the core ultrastructure was largely preserved after interacting with the glass powder.

Applied Biosciences doi: 10.3390/applbiosci5030058

Authors: Somya Sephalika Sanjoy Majumder Animesh Pattnaik Arpita Arsmika Sahu Nirmal Kumar Mohakud Bikash Ranjan Sahu

Pneumolysin (PLY), a cholesterol-dependent cytolysin produced by Streptococcus pneumoniae, plays a key role in the pathogenesis of bacterial pneumonia. The present study aimed to identify potential phytocompound inhibitors of pneumolysin and evaluate their protective effects against toxin-induced cytotoxicity using combined in silico and in vitro approaches. A ligand library comprising 200 phytocompounds was constructed using three-dimensional structures obtained from the PubChem database, while the PLY protein structure was retrieved from the Protein Data Bank. Molecular docking was performed to analyse protein-ligand interactions, followed by visualization using BIOVIA Discovery Studio Visualizer. Top-ranked phytocompounds from docking were further screened by computational ADME analysis, followed by molecular dynamics (MD) simulations for stability analysis. Selected compounds were then validated using RAW 264.7 macrophages, and cytotoxicity was assessed by flow cytometry. Among the screened compounds, three showed high binding affinity, with oridonin exhibiting the most favourable interaction profile (−7.906 kcal/mol). MD simulation confirmed the stability of the pneumolysin–oridonin complex. In vitro results demonstrated that pneumolysin induced significant, concentration-dependent cytotoxicity, whereas pre-incubation with oridonin significantly reduced cell death. To provide direct functional evidence of pneumolysin inhibition, haemolysis inhibition assay using sheep erythrocytes was performed where reduction of pneumolysin-mediated haemolysis by oridonin in a concentration-dependent manner was demonstrated. These findings suggest that oridonin may serve as a potential inhibitor of pneumolysin-mediated cytotoxicity and pneumococcal virulence.

Applied Biosciences doi: 10.3390/applbiosci5030057

Authors: Medhanie Mehari Johnstone O. Neondo Cecilia M. Mweu Tadesse Mehari Samuel G. Misginna

Potato (Solanum tuberosum L.) is a vital food security and cash crop in Eritrea, yet bacterial and fungal pathogens responsible for 15–30% yield losses remain molecularly uncharacterized across its production systems. Here, we present the first nationwide amplicon-based metagenomic survey of potato pathogen communities, sampling 81 farms across 14 sub-regions spanning four agroclimatic regions during July–August 2023. High-throughput amplicon sequencing targeting the bacterial 16S rRNA V3–V4 region and fungal ITS1–ITS2 loci revealed pronounced geographic heterogeneity in community composition and alpha diversity. Pseudomonas spp. were ubiquitous, with a peak relative abundance of 32.5% in Dekemhare. Dominant fungi included Alternaria spp. (14.3% in Berik), Fusarium spp. (highest diversity 53.8% in Adi Kuala), Botrytis cinerea (36.9% in Adi Keih), and Rhizoctonia solani (44.4% in Adi Tekeliezan). Bacterial Shannon diversity averaged 5.67; fungal, 4.70. Weighted UniFrac PCoA accounted for 56.5% of bacterial community variance along PC1, confirming distinct geographic clustering. Potential pathogen-associated taxa of Pseudomonas, Alternaria, Fusarium, and Colletotrichum were detected in every asymptomatic sample examined, demonstrating the inadequacy of visual-only disease surveillance. These findings establish the first molecular baseline for potato-associated taxon diversity in Eritrea, providing the empirical foundation for region-specific integrated disease management and evidence-based seed certification protocols.

Applied Biosciences doi: 10.3390/applbiosci5030056

Authors: Sara Reis Valentim Coelho Toufiq Yussif Rosalina Pereira Andreia Tomás Eugénia Gouveia

Chestnut brown rot caused by Gnomoniopsis smithogilvyi represents an increasing threat to Castanea sativa production, although its endophytic behaviour and response to pre-harvest management strategies under field conditions remain poorly understood. This study evaluated the occurrence of G. smithogilvyi in different chestnut tissues and assessed the effects of targeted field treatments on pathogen colonisation and fruit quality in commercial orchards located in north-eastern Portugal. Field trials included applications of a biological fungicide (Problad®), an inorganic micronutrient fertilizer (Fungicrops Bio®), a sulfur-micronutrient fertilizer (KSC Mix®), and a potassium-phosphonate chemical fungicide (Fosetyl-Al®), alongside untreated control orchards. Samples of leaves, branches, burrs, and nuts were subjected to microbiological and molecular analyses, while fruit external quality parameters were also assessed. G. smithogilvyi was detected in all analysed organs, confirming its widespread endophytic occurrence. Treatment effects were more pronounced in reproductive tissues, with Problad® and Fosetyl-Al® significantly reducing pathogen incidence in burrs and nuts compared with the control. KSC Mix® promoted fruit development but did not significantly reduce fungal incidence. Treated orchards generally showed improved fruit quality and lower insect infestation. Phylogenetic analysis confirmed the identity of all isolates as G. smithogilvyi. Overall, the results highlight the complexity of managing chestnut brown rot and support the need for integrated and sustainable disease management strategies.

Applied Biosciences doi: 10.3390/applbiosci5030055

Authors: Luckas Obanda Malachy Betty Schwartz Natalie Avital-Cohen Ofer Gover Hadar Bar-Dagan Shelly Druyan Joanna Bartman Asaf Marco Dekel Tsalik Israel Rozenboim

The global push to eliminate antibiotic growth promoters in poultry has accelerated the demand for effective natural alternatives. β-Glucans—branched polysaccharides derived from Saccharomyces cerevisiae cell walls—enhance immunity and gut health; however, their mechanistic effect on the somatotropic axis and meat quality in broilers remains unresolved. Herein, the hypothesis that dietary β-glucan modulates somatotropic signaling to improve growth performance and breast muscle quality was tested with 240 one-day-old Ross 308 chicks allocated to three groups—untreated control, 250 mg β-glucan/kg feed, and 1 g β-glucan/kg feed—and reared for 35 d. Growth performance, plasma growth hormone (GH) and prolactin (PRL), somatotropic axis gene expression in liver and breast muscle, and postmortem meat quality were assessed. β-Glucan supplementation significantly elevated final body weight, breast muscle weight, and plasma GH and PRL, and upregulated hepatic IGF-1 and muscle GH receptor mRNA at 35 d, and hepatic GH receptor mRNA at 17 d. Muscle pH was higher and relative drip loss lower in supplemented birds 72 h postmortem. These results support the hypothesis and identify 1 g β-glucan/kg feed as an effective dose for improving growth and meat quality through somatotropic axis modulation—a novel mechanistic demonstration in broiler chickens.

Applied Biosciences doi: 10.3390/applbiosci5030054

Authors: Angel David Hernández-Amasifuen Julio Cesar Santos-Pelaez Jheyson Yopan-De la Cruz Jorge Alberto Condori-Apfata Juan Carlos Guerrero-Abad

Extracting high-molecular-weight (HMW) DNA from cactus tissues remains technically challenging due to the abundance of mucilage, pectins, polyphenols, and other metabolites that compromise DNA purity, increase viscosity, and reduce integrity, thereby limiting its suitability for long-read sequencing. This constraint is particularly relevant in Selenicereus megalanthus, a crop of increasing agronomic and genomic importance for which optimized protocols for third-generation sequencing remain limited. Here, we compared four CTAB-based DNA extraction protocols using dehydrated cladode tissue and evaluated DNA quality using NanoDrop spectrophotometry, Qubit fluorometry, agarose gel electrophoresis, and functional validation via sequencing on the Oxford Nanopore PromethION 2 Solo platform. Among the tested methods, our proposed optimized mucilage-adapted CTAB (MuCTAB) protocol, comprising 4% CTAB, 4% PVP-40, 0.5% β-mercaptoethanol, and proteinase K, showed the best overall performance. MuCTAB yielded the highest dsDNA concentration (239.63 ± 34.37 ng/µL), optimal purity ratios (A260/A280 = 1.96 ± 0.05; A260/A230 = 2.01 ± 0.01), and superior DNA integrity. Nanopore validation confirmed its effectiveness, with MuCTAB producing the highest sequencing yield (84.2 Gbp), read count, mean read quality score (Q18.4), read N50 (40.3 kbp), and maximum read length (1.9 Mbp). Overall, MuCTAB represents a low-cost, reproducible, and efficient method for HMW DNA extraction from mucilage-rich pitahaya tissues and shows promising potential for adaptation to other recalcitrant plant species.

Applied Biosciences doi: 10.3390/applbiosci5030053

Authors: Hadeel Mazin Akram Saif Sehaam Saliem

Periodontitis is a chronic inflammatory disease initiated by dysbiotic biofilms and sustained by an altered host response, resulting in progressive destruction of the tooth-supporting tissues. Alongside immune-mediated damage, epithelial–mesenchymal transition has been proposed as a contributing mechanism in gingival barrier disruption and periodontal tissue remodeling. This narrative review examines the roles of the EMT-related transcription factors Snail1 and Twist1 in periodontitis, with particular focus on their involvement in epithelial junction loss, mesenchymal activation, and disease-associated tissue change. Relevant literature was identified through targeted searches of PubMed/MEDLINE and Google Scholar using terms related to periodontitis, gingival epithelium, EMT, Snail, and Twist, while selected non-periodontal studies were used only for mechanistic context where direct evidence was limited. Current findings suggest that periodontal disease is commonly associated with reduced epithelial adhesion markers and increased mesenchymal-associated markers, with Snail1 appearing more closely linked to epithelial repression and Twist1 to motility and matrix remodeling. However, the evidence remains largely preclinical or associative. Overall, Snail1 and Twist1 may participate in EMT-like remodeling in periodontitis, but periodontal-specific mechanistic and functional studies are still needed to clarify causality and clinical relevance.

Applied Biosciences doi: 10.3390/applbiosci5030052

Authors: Thi My Linh Dao Viet The Ho

Biocontrol using antagonistic bacteria is considered a promising and sustainable approach for managing tomato bacterial diseases. Many antagonistic bacterial strains show strong activity under laboratory conditions, but their performance in greenhouse and field conditions is often unstable. This discrepancy is commonly described as the “translation gap”, meaning the gap between laboratory/greenhouse success and inconsistent field performance. This review examines the main causes of this gap. The analysis indicates that inconsistent field performance is not caused by a single factor, but by the combined effects of limited ecological fitness, environmental variability, formulation and delivery problems, and differences in agricultural management practices. Laboratory and greenhouse systems often simplify plant–bacteria–pathogen interactions and do not fully reflect the complexity of field environments. As a result, they may overestimate the practical potential of bacterial biocontrol agents. A conceptual framework is proposed to link biological, environmental, technical, and operational constraints and to explain why bacterial biocontrol often fails under practical conditions. Special attention is given to tropical tomato production systems, with emphasis on Vietnam, where high temperature, rainfall, humidity, intensive cultivation, and limited field-based evidence further increase the difficulty of achieving consistent disease control. Overall, this review highlights the need to move beyond simple antagonistic activity-based screening and to give greater attention to ecological fitness, rhizosphere colonization, stress tolerance, formulation quality, and compatibility with local farming practices.

Applied Biosciences doi: 10.3390/applbiosci5020051

Authors: Midori Kurahashi Angelica Naka Hiroyuki Ishiwata Tadashi Shoji

Filamentous cyanobacteria in the genus Nostoc show color diversity in their natural habitats, ranging from bright green to yellow–green to brown. In contrast, laboratory cultures are typically uniform in color, which is attributed to differences in light spectrum and intensity. Nostoc contains several bioactive pigments, including phycocyanin, phycoerythrin, allophycocyanin, carotenoids, and scytonemin, which are involved in light harvesting, photoprotection, and UV screening. Variations in light spectrum and intensity regulate the abundance and organization of these pigments, altering colony coloration and affecting photosynthetic performance, stress tolerance, and growth. These changes impact biomass and pigment productivity. In this review, we synthesize recent findings on how light regulates pigment biosynthesis in Nostoc and examine how wavelength, irradiance, and photoperiod influence pigment composition, biomass accumulation, and pigment productivity, considering both underlying photoregulatory mechanisms and cultivation design. This perspective connects photophysiological insights with practical strategies, including staged lighting and LED-based systems, to improve pigment yield while maintaining biomass production. These insights support the future application of Nostoc pigments in food, cosmetic, and pharmaceutical sectors.

Applied Biosciences doi: 10.3390/applbiosci5020050

Authors: Liviu Marian Bogdan Daniel Berean Stefan Coman Ionela Ut Simona Ciupe Raluca Cimpean

Reproductive efficiency is a critical determinant of productivity and profitability in both dairy and beef cattle systems. Progesterone-based estrus synchronization protocols have emerged as essential tools to control the timing of ovulation, enhance artificial insemination efficiency, and optimize herd reproductive performance. These protocols exploit the physiological regulation of the hypothalamic–pituitary–ovarian axis, maintaining elevated progesterone levels to suppress ovulation and coordinating luteolysis and follicular wave emergence through prostaglandins, GnRH analogues, and, in some cases, equine chorionic gonadotropin. In beef cattle, progesterone supplementation facilitates fixed-time artificial insemination, shortens calving intervals, and improves calf crop uniformity, whereas in dairy cows it mitigates poor estrus detection, enhances service rates, and reduces days open. Fertility outcomes are influenced by cow physiological status, metabolic condition, and protocol adherence, with pregnancy per AI typically ranging from moderate to high in beef cattle and lower in lactating dairy cows. Economic evaluation indicates that these protocols are cost-effective in herds with moderate to large size, low estrus detection efficiency, and intensive management, although labor, drug costs, and handling requirements must be considered. Critical analysis suggests that synchronization cannot compensate for underlying nutritional, health, or metabolic deficiencies, and future research should focus on precision application, individualized protocols, and integration with automated reproductive monitoring to improve both biological and economic efficiency. This review summarizes physiological principles, protocol designs, practical applications, fertility outcomes, and economic considerations of progesterone-based estrus synchronization in cows, highlighting current challenges and perspectives for enhanced reproductive management.

Applied Biosciences doi: 10.3390/applbiosci5020049

Authors: Dwi Anita Suryandari Aryo Tedjo Puji Budi Setia Asih Din Syafruddin Fadilah Fadilah

Background: Severe malaria remains a major cause of morbidity and mortality, yet the conserved molecular signatures underlying complicated infections across Plasmodium vivax (P. vivax) and Plasmodium falciparum (P. falciparum) are not well characterized. Identifying shared transcriptional biomarkers and host–parasite interaction networks is crucial for improving diagnosis and discovering new therapeutic targets. Methods: Public transcriptomic datasets (GSE55644, GSE59844, GSE34404) were analyzed using GEO2R to identify differentially expressed genes (DEGs). Volcano plots, Venn diagrams, and KEGG mapping were used to identify conserved DEGs. Principal Component Analysis (PCA) and Support Vector Machine (SVM) models were used to assess predictive performance. Host–parasite cross-species correlation analysis integrated parasite DEGs with host hub-genes. Functional enrichment and network module analysis were performed using Cytoscape v3.10.2 and GO/KEGG annotation tools. Results: A total of 3363 DEGs were identified in P. vivax (GSE55644) and only one DEG in P. falciparum (GSE59844) using adjusted p-values, though 772 DEGs emerged with unadjusted p-values. Cross-dataset comparison revealed 18 common DEGs, with eight upregulated genes—TIM9, NUF2, SRP68, HDAC1, GRP94, DHHC8, PPM9, and RPL27—showing robust predictive performance (AUC = 1.000; CA = 1.000) for distinguishing complicated from uncomplicated malaria in both species. Host analysis identified 1719 DEGs and six hub-genes (TNF, IL6, TLR4, CR1, CD40LG, ICAM1) linked to apoptosis, Toll-like receptor signaling, complement cascades, and cell adhesion. SVM validation predicted parasitemia levels with 75.5–84.0% accuracy. Cross-species correlation revealed strong positive interactions between parasite HDAC1/GRP94 and host IL6/TNF and negative correlations involving NUF2, TIM9, ICAM1, and CR1. Functional enrichment analysis highlighted ER stress, immune activation, and erythrocyte adhesion pathways, which together form three major host–parasite modules. Conclusion: These findings highlight conserved biomarkers and potential therapeutic candidates for future validation, demonstrating that combined DEG profiling and machine-learning approaches can provide a powerful framework for improving diagnostics and intervention strategies for severe malaria.

Applied Biosciences doi: 10.3390/applbiosci5020048

Authors: Angel David Hernández-Amasifuen Diego Hiroshi Takei-Idiaquez Flor Matilda Yupanqui-Morales Alexandra Jherina Pineda-Lázaro Juan Carlos Guerrero-Abad

One of the major agricultural, nutritional, and medicinal resource in the plant kingdom is the family of Cucurbitaceae, which is also recognized for its richness in carotenoids, terpenoids and triterpenoids. Mevalonate diphosphate decarboxylase (MVD) plays a crucial role in the mevalonate pathway by catalyzing a key step in isoprenoid biosynthesis, which is important for plant growth as well as for responses to biotic and abiotic stresses. Despite its metabolic relevance, comparative analyses of the MVD gene and protein in cucurbits remain limited. Therefore, this study aimed to identify and characterize MVD gene and protein in Cucurbitaceae using in silico approaches. Homology searches, multiple sequence alignment, phylogenetic and selective pressure analyses, physicochemical characterization, structural prediction, conserved motif analysis, cis-regulatory element prediction, and public expression profiling were performed. The predicted proteins showed high conservation in amino acid sequence, motif organization, and structural conformation, with lengths ranging from 398 to 424 aa. Descriptive FPKM-based transcriptomic profiles in Cucumis sativus showed higher MVD expression values in reproductive tissues and an apparent increase under powdery mildew infection. These findings suggest a potential role of the MVD gene in the Cucurbitaceae family and provide an exploratory framework for future studies on terpenoid and triterpenoid metabolism, promoter regulation, and stress-associated transcriptional responses.

Applied Biosciences doi: 10.3390/applbiosci5020047

Authors: Ali Eskandari Xiaoling Kang Marc G. Aucoin D. Moira Glerum Ting Y. Tsui

As the use of electronics and mobile devices increases, indium and its related compounds are increasingly prevalent in consumer products. However, the effects of the ionic form of indium on the mammalian renal cells are unclear. Understanding indium toxicity in these cells is important, as it relates to kidney health. Kidneys remove heavy metals, maintain electrolyte balance, and perform other vital functions. This in vitro study examines the effects of indium chloride (InCl3) on Vero cells, focusing on cell morphology, viability, reactive oxygen species (ROS) production, and expression of key focal adhesion proteins. Cells were incubated in culture media with InCl3 concentrations ranging from 0 to 3.2 mM for 24 h. Fluorescence confocal microscopy analyses revealed that concentrations above 0.8 mM caused the cells to become more compact and display decreased actin filament lengths, suggesting cellular degeneration, which was further supported by the AlamarBlue® Cell Viability Reagent. Using a 2′,7′–dichlorofluorescin diacetate (DCFDA/H2DCFDA) assay, we show that ROS levels increase with InCl3 concentration, accompanied by significant increases in focal adhesion kinase (FAK) and paxillin at InCl3 concentrations above 0.8 mM. Interestingly, the level of α-actinin detected is not affected by exposure to InCl3. Our findings demonstrate that InCl3 has negative impacts on the growth and behaviour of Vero cells at concentrations exceeding 0.8 mM, underscoring the need for further investigation into the biological effects of indium-containing compounds.

Applied Biosciences doi: 10.3390/applbiosci5020046

Authors: Edwis R. García-Villavicencio Eduardo Lira-Díaz Armando Sunny César Díaz-Talamantes Cristina Burrola-Aguilar María Elena Estrada-Zúñiga Carmen Zepeda-Gómez Juan J. Acevedo Fernández Gabriela Castañeda-Corral Raúl A. Morales-Luckie María G. González-Pedroza

Green synthesis of nanoparticles using biological extracts has emerged as a sustainable alternative to conventional physicochemical methods, as it provides eco-friendly reducing and stabilizing agents. In this study, silver–copper bimetallic nanoparticles (Ag/Cu-BNPs) were biosynthesized using an aqueous extract of the medicinal mushroom Hericium erinaceus, and their biological properties were evaluated. Three nanoparticle formulations (M1–M3), differing only in the proportion of H. erinaceus extract, were analyzed and selected based on their optical response in UV–Vis spectroscopy. The synthesized nanoparticles were characterized by UV–Vis spectroscopy, FTIR, TEM, and EDS. Antibacterial activity was assessed against Escherichia coli and Staphylococcus aureus using standard methods, while cytotoxicity was evaluated in cancer cell lines (Caco-2, HT-29, A549, MCF-7) and a normal fibroblast line (HDFn). The synthesized Ag/Cu-BNPs were quasi-spherical with an average size of 9.3 ± 3.4 nm, calculated from TEM image analysis of over 900 particles. They exhibited significant antibacterial activity, particularly against S. aureus, and showed dose-dependent cytotoxicity, with Caco-2 cells being the most sensitive. Notably, the nanoparticles displayed greater selectivity toward cancer cells compared to normal fibroblasts, suggesting potential biomedical applications in antimicrobial and antitumor therapies. These results suggest that H. erinaceus extract is an effective biogenic agent for Ag/Cu-BNP synthesis, and that the resulting nanoparticles possess antimicrobial and cytotoxic properties that warrant further investigation for potential biomedical applications.

Applied Biosciences doi: 10.3390/applbiosci5020045

Authors: Dody Novrial Nor Sri Inayati Nur Signa Aini Gumilas Dhadhang Wahyu Kurniawan Sutiman Bambang Sumitro

Objective: The development of nanobubble technology has expanded to various gas molecules, such as nitric oxide (NO). NO donors have been frequently used for both therapeutic and diagnostic purposes. However, the use of NO in nanoformulations has received little attention thus far. This research aimed to assess the subchronic toxicity of NO nanobubbles in a preclinical study design. Methods: Ninety days of subchronic toxicity testing based on modified OECD guidelines in Sprague Dawley rats was used in this study to assess potential adverse effects of intravenous administration of NO nanobubbles (NONBs). Hematological factors, serum biochemistry, and histology of the liver, kidney, heart, lung, and spleen were investigated. Results: The intravenous NONB injection trial using graded doses of 0.01 mL, 0.04 mL, and 0.06 mL resulted in no deaths during the 90-day treatment period. However, liver disturbance and electrolyte imbalance emerged. This condition was supported by histological findings in the liver and kidney, which showed potential reversible damage, and persistent hemorrhage of the spleen. Conclusions: Intravenous administration of NONBs at a dose of up to 0.06 mL in this subchronic toxicity test remains safe. Further studies with adjustments to the test formulation are highly encouraged before the clinical trial stage in humans.

Applied Biosciences doi: 10.3390/applbiosci5020044

Authors: Anastasia S. Tugbaeva Gregory I. Shiryaev Mohamad Darkazanli Olga V. Voropaeva Ekaterina E. Ryabova Alexander A. Ermoshin Galina G. Borisova Maria G. Maleva Irina S. Kiseleva

Salinization of agricultural soils is a global problem causing crop yield declines. This impact is caused by osmotic and oxidative stress, which plants often rely on endophytic bacteria to overcome. A bacterial isolate from the roots of the halophyte Plantago salsa was studied between 2024 and 2026, and its ability to increase barley tolerance to moderate salt stress was determined. Based on 16S rRNA gene sequencing (1410 bp), the isolate PS-50.1 was identified as Providencia sp. It demonstrated key plant growth-promoting properties, including indole-3-acetic acid production (21.4 mg L−1) and phosphate solubilization (69.0 mg L−1). The strain supported barley growth at 7% NaCl. Inoculation of barley seeds with this strain (108 CFU L−1) significantly reduced moderate salt stress in plants both in vitro and in a pot experiment. Inoculated plants under salinity conditions had greater shoot length (+11.6%) compared to non-inoculated; higher pre-flag leaf fresh weight; demonstrated decreased levels of prooxidants (H2O2 by 44.8% and malondialdehyde by 31.8%), higher proline accumulation (up to 2.0-fold), and increased antioxidant enzyme activity (catalase by 26.6% and ascorbate peroxidase by 191%). Furthermore, inoculated plants showed 9.4% higher water use efficiency and photosynthetic rate (+5.5%) under salt stress compared to uninoculated plants. These results indicate that the halophytic strain Providencia sp. PS-50.1 is a promising candidate for the development of microbial preparations aimed at increasing crop productivity under saline conditions.

Applied Biosciences doi: 10.3390/applbiosci5020043

Authors: Dimas Rahadian Aji Muhammad Marliana Bella Saputri As-salamah Karomatusy Syifa Danar Praseptiangga Setyaningrum Ariviani

This study evaluated the aroma and antioxidant properties of single-origin chocolate produced from cocoa beans sourced from seven regions in Indonesia. Two roasting approaches—dynamic and static—were applied, followed by small-scale chocolate processing using a melanger. Both raw cocoa beans and resulting chocolates were comprehensively characterised to examine relationships between raw material quality and final product attributes. The results show that the cocoa beans exhibited significant regional variation in physicochemical properties, including moisture, protein, pH, phenolic and flavonoid contents, antioxidant activity, and the fermentation index. These differences were reflected in the chocolates, which showed distinct appearance, aroma profiles, and antioxidant capacities. Roasting methods significantly influenced moisture content, colour, and antioxidant properties, with static roasting yielding chocolates with higher moisture, greater antioxidant retention, and lighter colour compared to dynamic roasting. Gas chromatography–mass spectrometry (GC–MS) analysis identified key volatile compounds such as acetic acid, 2-phenylethyl acetate, phenylethyl alcohol, acetoin, and pyranone as dominant contributors to aroma. Regional origin and processing conditions play critical roles in defining the quality of a single-origin chocolate. The knowledge obtained in this study can support the chocolate industry by enabling raw material selection, optimising roasting strategies, and standardising processes to achieve targeted quality attributes.

Applied Biosciences doi: 10.3390/applbiosci5020042

Authors: Nandita Das Rajib Mondal Ujjwal Layek Prakash Karmakar

Identifying the nectar sources of stingless bees is essential for understanding plant–pollinator interactions and for promoting sustainable meliponiculture. Traditionally, this has been achieved through melissopalynological analysis of honey; however, this approach has certain limitations. Here, we aimed to evaluate whether pollen analysis of bee crop nectar can reliably identify nectar sources for stingless bees. We conducted palynological analyses of honey samples (n = 12) and crop nectar samples (n = 757, considering individual foragers), and conducted field surveys to determine nectar sources for the stingless bee Tetragonula pagdeni in West Bengal, India. From the honey analysis, 42 pollen types were identified, with Eucalyptus tereticornis as the predominant pollen type. In contrast, 67 pollen types were recorded from the crop nectar samples. The most frequently occurring pollen types were Acacia auriculiformis, Borassus flabellifer, Eucalyptus tereticornis, Peltophorum pterocarpum, Tridax procumbens, and Ziziphus mauritiana. Through field surveys, 73 plant species were identified as nectar sources. By integrating these methods, 85 plant species were recognised as nectar sources for stingless bees. The findings indicate that palynological analysis of bees’ crop nectar is an effective method for identifying the nectar sources of a bee species. Furthermore, combining palynological analysis of crop nectar with melissopalynological analysis of honey provides a more comprehensive and potentially more accurate assessment of nectar sources than reliance on honey analysis alone.

Applied Biosciences doi: 10.3390/applbiosci5020041

Authors: Agustyas Tjiptaningrum Yusra Yusra Kusmardi Kusmardi Ade Arsianti Sjahjenny Mustokoweni Fadilah Fadilah

Background: Cytokine storm-like inflammation includes an imbalanced immune response, where excessive interleukin-6 (IL-6) and inadequate IL-10 play a central role in increasing tissue injury. Melaleuca cajuputi leaves are known to contain anti-inflammatory bioactive compounds. However, the potential to modulate the dysregulated cytokine response remains underexplored. Objective: This study aimed to evaluate the immunomodulatory effects of Melaleuca cajuputi subsp. cajuputi Powell Ethanolic Leaf Extract (MC-ELE) on IL-6, IL-6R, and IL-10 levels in a BALB/c mouse model of lung inflammation induced by lipopolysaccharide (LPS). Methods: Phytochemical screening was performed to identify active constituents in MC-ELE. Male BALB/c mice were intratracheally challenged with LPS (mg·kg−1 BW) to induce cytokine storm-like inflammation. After 24 h, mice received oral MC-ELE at doses of 750, 1500, 3000 mg·kg−1 BW, or dexamethasone (10 mg·kg−1 BW), for seven consecutive days. On day eight, serum and bronchoalveolar lavage fluid (BALF) were collected for IL-6, IL-6R, and IL-10 assessment using ELISA. Furthermore, body weight changes and clinical symptoms were monitored throughout the study. Results: MC-ELE was confirmed to contain anti-inflammatory compounds. Across all groups, IL-6 concentrations in BALF were consistently higher than in serum, with the LPS-only group showing the greatest elevation. Serum IL-6R levels exceeded BALF IL-6R levels in most groups, except at 1500 mg·kg−1 BW MC-ELE dose. BALF IL-10 was higher compared with serum in all MC-ELE-treated groups. Therefore, MC-ELE might preferentially enhance anti-inflammatory responses within the pulmonary microenvironment. There was no observed toxicity or weight loss at doses up to 3000 mg·kg−1 BW. Conclusions: MC-ELE reported promising immunomodulatory activity by lowering IL-6 and IL-6R levels while enhancing IL-10 responses in lung inflammation induced by LPS within lung tissue. These results suggested its potential as a natural therapeutic candidate for managing severe inflammatory conditions.

Applied Biosciences doi: 10.3390/applbiosci5020040

Authors: Amalia Despoina Koutsogianni Georgios Tatsis Christina Telli Konstantinos Stamoulis Haralampos Milionis Evangelos Liberopoulos Alexandros D. Tselepis Constantinos Tellis

Low levels of high-density lipoprotein cholesterol (HDL-C) are strongly associated with increased cardiovascular risk. However, under various pathological conditions, high-density lipoprotein (HDL) particles may undergo structural and functional modifications, leading to a progressive loss of antioxidant capacity and a shift from a cardioprotective to a proatherogenic phenotype. In this cross-sectional study, we investigated differences in HDL particle distribution and antioxidant function between individuals with elevated lipoprotein(a) [Lp(a)] levels (≥30 mg/dL) and those with low Lp(a) levels (<10 mg/dL). Serum low-density lipoprotein (LDL) and HDL subfractions were analyzed in twenty subjects with high Lp(a) and ten low-Lp(a) controls using non-denaturing polyacrylamide gel electrophoresis (PAGE, Lipoprint system). Enzymatic activities of paraoxonase-1 (PON1) and HDL-associated lipoprotein-associated phospholipase A2 (HDL-Lp-PLA2) were measured. Electrophoretic analysis revealed a significant increase in small HDL (S-HDL) in the high-Lp(a) group compared to the controls (34.1 ± 13.2% vs. 21.5 ± 2.7%, p = 0.01), alongside a reduction in large HDL (L-HDL) (19.6 ± 9.9% vs. 33.4 ± 3.8%, p < 0.001). Furthermore, the high Lp(a) group exhibited significantly lower HDL-PON1 activity (55 ± 12 vs. 67 ± 7 U/mL, p < 0.001) and HDL-Lp-PLA2 activity (2.6 ± 1.0 vs. 3.6 ± 1.2 nmol/min/mL, p < 0.02) compared with the controls. These findings suggest that markedly elevated Lp(a) levels are associated with a shift toward a more proatherogenic HDL subfraction profile and impaired antioxidant functionality, which may reflect mechanisms linked to increased atherosclerotic cardiovascular disease (ASCVD) risk.

Applied Biosciences doi: 10.3390/applbiosci5020039

Authors: Laia L. Fernández Carmen Bedia Joaquim Jaumot

Glyphosate-based herbicides are widely used in agriculture. However, their broader effects on plant lipid metabolism remain insufficiently characterized beyond their canonical target, the shikimate pathway. In this study, we evaluated tissue-specific lipidomic responses of carrot (Daucus carota L.) plants grown under controlled conditions following sublethal foliar exposure to a commercial glyphosate-based herbicide formulation. Leaves, leaf stalks, and roots were harvested 30 days after application, and lipid extracts were analyzed using ultra-high-performance liquid chromatography coupled to mass spectrometry. Multivariate statistical analyses were applied to assess treatment-related differences. Morphological parameters showed no major visible symptoms, although minor changes in shoot architecture were observed. Untargeted lipidomic profiling revealed treatment-associated, tissue-specific alterations in lipid composition. In leaves, changes were detected in free fatty acids, tocopherols, and galactolipids, whereas leaf stalks and storage roots showed alterations mainly affecting phospholipid and glycerolipid classes. In summary, lipid profiles indicated shifts in the relative abundance of membrane- and storage-related lipid species. These results suggest that sublethal exposure to a glyphosate-based herbicide formulation may be associated with measurable lipidomic differences in carrot tissues, highlighting the sensitivity of untargeted lipidomic profiling for detecting metabolic responses to agrochemical exposure.

Applied Biosciences doi: 10.3390/applbiosci5020038

Authors: Hayden D. Hedman

This study presents an exploratory reinforcement learning (RL)-based simulation framework for examining antimicrobial resistance (AMR) dynamics under repeated exposure to a non-antibiotic stressor, using copper as a simplified model compound. The objective is not to provide mechanistic or predictive insight into microbial evolution, but to evaluate how alternative sequential decision-making strategies perform within a constrained and transparent simulation environment. Three agent strategies were compared: random action selection, a rule-based heuristic, and a tabular Q-learning agent. Simulations were conducted over fixed 40-cycle episodes in which agents adjusted copper exposure in response to evolving resistance-related state variables. Across experimental runs, the Q-learning agent exhibited lower cumulative resistance burden, measured by area under the curve (AUC) of minimum inhibitory concentration (MIC) trajectories for chloramphenicol and polymyxin B, while maintaining lower cumulative copper exposure relative to baseline strategies. The rule-based agent demonstrated intermediate performance, whereas the random agent showed greater variability and less stable trajectories. These findings reflect differences in simulated control behavior within a simplified stochastic system. Overall, this work introduces an interpretable reinforcement learning simulation tool intended to support comparative evaluation of adaptive versus static strategies in antimicrobial pressure management under limited observability.

Applied Biosciences doi: 10.3390/applbiosci5020037

Authors: Lorenzo Cipriano

Molecular diagnosis has advanced rapidly in neurogenetic disorders, yet translating genotype into patient-specific predictions of brain network dysfunction and progression remains limited. Virtual brain models provide a structured solution by embedding individual anatomy and connectomics into biophysical whole-brain simulations. The critical step is to position genetics not as a diagnostic label, but as a constructive input to model design. This review outlines a genetics-centered framework for virtual brain modeling. First, atlas-derived transcriptomic and cell-type maps can define region-specific molecular priors, constraining vulnerability or excitability parameters and reducing model degeneracy. Second, when reproducible genotype-linked network phenotypes exist, mutation groups can inform stratified initialization and progression regimes. Third, at the patient level, exome and CNV data—summarized as pathway burdens and, where appropriate, calibrated polygenic modifiers—can be translated into individualized priors or regularizers, provided that mapping rules are explicit and externally validated. By integrating genetics at multiple levels of evidence, virtual brain models gain mechanistic plausibility, improved calibration, and explicit uncertainty quantification. The most realistic impact over the next few years is likely to be improved stratification, progression-aware forecasting, and scenario-based decision support in rare neurogenetic diseases, especially where longitudinal cohort infrastructure and validated biomarker inputs are already available, rather than deterministic individual prediction.

Applied Biosciences doi: 10.3390/applbiosci5020036

Authors: Victoria K. Mwaeni Dorothy Nyamai Samoel A. Khamadi Sophia K. Musenjeri Hellen Kariuki Mutinda Cleophas Kyama

Cervical cancer is associated with persistent human papillomavirus (HPV) infections. The early detection of HPV is one of the key strategies for the effective treatment of cervical cancer. Current HPV molecular detection methods use enzyme-based nucleic acid amplification strategies that, although specific and sensitive, involve extensive workflows. Enzyme-free isothermal amplification detection strategies with the potential to adapt to low-resource settings for HPV oncogenic transcripts remain limited. This study aimed to validate a fluorescence-based branched hybridization chain reaction (bHCR) assay for the targeted detection of HPV 16/35 E6 oncogenic transcripts. Analytical performance was evaluated using a synthetic target and a negative clinical matrix, whereas the diagnostic performance of the bHCR assay was evaluated using clinically characterized samples (n = 67). The study demonstrated assay linearity over an analyte concentration range of 0.625–40 µM, with a statistically significant correlation between the fluorescence signal and target concentration (r2 = 0.928, p < 0.0001). Analytical accuracy was assessed by pre-extraction spike recovery; achieved recoveries ranged from 70% to 86%, indicating potential RNA loss during the assay workflow. Analytical sensitivity determined the background signal threshold limit of blank (LoB) as 16,251.6 RFU, with detection and quantification at concentrations of 0.0625 µM (≈2.6 × 1011 copies per reaction, limit of detection (LoD) and 0.125 µM (≈5.3 × 1011 copies per reaction, limit of quantification (LoQ). The assay exhibited high diagnostic performance, with a diagnostic cut-off of 16,481 RFU and an area under the curve (AUC) of 0.9194. Specificity and sensitivity of the assay were 94% and 86%, respectively, with a Negative Predictive Value (NPV) of 85% and a Positive Predictive Value (PPV) of 94%. These findings demonstrate a reliable analytical assay with excellent diagnostic discrimination and warrant further optimization and expanded clinical validation.

Applied Biosciences doi: 10.3390/applbiosci5020035

Authors: Daniel Berean Liviu Marian Bogdan Simona Ciupe Raluca Cimpean

Efficient reproductive management is essential for optimizing productivity and sustainability in sheep and goat production systems. Estrus synchronization (ES) has emerged as a pivotal tool for coordinating mating, enhancing fertility, facilitating artificial insemination (AI), and supporting out-of-season breeding. Hormonal protocols, including progesterone devices, prostaglandins, and gonadotropin or gonadoliberine treatments, provide the highest precision in estrus and ovulation timing, with estrus response rates exceeding 90% and conception rates commonly between 65–85%. These methods are particularly effective in intensive or AI-based systems but are constrained by cost, labor, regulatory restrictions, and welfare considerations. Non-hormonal strategies, such as the ram effect, photoperiod manipulation, nutritional flushing, and management-based interventions, exploit natural physiological, socio sexual, and nutritional cues to partially synchronize estrus. While these approaches exhibit greater variability and lower precision than hormonal methods, they offer advantages in low input, organic, and extensive systems by improving reproductive clustering, ovulation, and lambing compactness. Among these, the ram effect is the most effective and widely applicable. Integrated reproductive management, combining hormonal or non-hormonal strategies with optimized nutrition, health, and flock management, is critical for achieving predictable and sustainable reproductive outcomes. Future research should focus on refining hormone-sparing protocols and enhancing the reliability of natural synchronization methods.

Applied Biosciences doi: 10.3390/applbiosci5020034

Authors: Anastasios Nikolopoulos Vangelis D. Karalis

Artificial intelligence has evolved to generative AI (GenAI), a paradigm shift that has shifted the emphasis away from the evaluation of existing patterns to the generation of novel biological and medical material. This study examines GenAI achievements in biosciences and medical fields the last five years in these fields using databases such as PubMed and Scopus. The paper highlights the recent evolution in biomedical research from virtual screening to de novo design. It illustrates how models like RFdiffusion and ProteinMPNN leverage “inverse folding” to assemble novel of proteins and drugs. Ultimately, these generative methods yield candidate with enhanced binding affinity and structural stability. For example, exploratory studies suggest GenAI has the potential to address inefficiencies via automatic documentation in the therapeutic sector, and it may enhance research capabilities by using Generative Adversarial Networks (GANs) and Variational Autoencoders (VAEs) to generate synthetic clinical trial data that preserves confidentiality. In addition, the review argues that though GenAI democratizes medical education through scalable simulations, it raises questions about long-term knowledge retention. Finally, GenAI also offers a transformative “write” capability for biology, but its responsible application will require addressing model “hallucinations” and building Explainable AI (XAI) and robust ethical frameworks.

Applied Biosciences doi: 10.3390/applbiosci5020033

Authors: Ana-Marija Gotal Skoko Ivana Flanjak Dajana Gašo-Sokač Martina Skendrović Babojelić Bojan Šarkanj Ivana Tomac Valentina Obradović Ante Lončarić

This study compared the quality and bioactive composition of cloudy apple juices produced from four traditional and four conventional apple cultivars immediately after harvest and following cold storage of the fruit at 4 °C for three and six months. Apples were harvested at the ripening stage at the same criteria, stored as whole fruit, and processed into cloudy juice after harvest, three, and six months of storage. Physicochemical parameters and sugar composition were determined, while phenolic compounds were quantified by HPLC-PDA. Antioxidant activity, total phenolic, and flavonoid content were measured spectrophotometrically. All analyses were performed in technical triplicate. The results revealed notable differences between traditional and conventional cultivars. Juices produced from traditional apple cultivars exhibited significantly higher total polyphenol and flavonoid contents than those from conventional cultivars. Significant variations in catechin, myricetin, quercetin, and epigallocatechin levels were also observed among cultivars. The traditional apple cultivar ‘Mašanka’ showed higher concentrations of quercetin (0.09 ± 0.01 µg/mL), chlorogenic acid (486.58 ± 5.48 µg/mL), catechin (8.76 ± 0.54 µg/mL), epicatechin (20.22 ± 0.20 µg/mL), and phloridzin (13.48 ± 0.19 µg/mL) compared to the other cultivars. In contrast, conventional cultivars showed higher concentrations of myricetin and procyanidin B1. Moreover, the content of TA, sucrose, and glucose decreased, whereas pH, fructose, TSS (except for ‘Fuji’ and ‘Granny Smith’) increased. The TFC decreased in traditional apple cultivars, while it increased in conventional cultivars; however, the TFC in conventional cultivars remained lower than in traditional ones. Overall, these findings demonstrate that the cold storage of apples significantly affects juice composition and highlight the advantages of traditional apple cultivars for producing juices with enhanced phenolic content and antioxidant activity.

Applied Biosciences doi: 10.3390/applbiosci5020032

Authors: Liu Xi Faez Firdaus Abdullah Jesse Bura Thlama Paul Eric Lim Teik Chung Mohd Azmi Mohd Lila

Brucellosis poses a persistent threat to livestock health in high-altitude pastoral regions of China, where harsh environments and semi-nomadic grazing increase transmission risk. Existing surveillance systems rely mainly on periodic serological testing and lack effective early warning capability. This study proposes an ensemble learning-based early warning framework integrating veterinary epidemiological indicators with environmental and herd-movement data. A total of 4826 herd-level records collected over five years (2019–2024) were analyzed, with an overall positivity rate of 11.4%. Multi-source data, including serological, clinical, reproductive, vaccination, meteorological, pasture-management, and herd-movement information (from GPS tracking and structured surveys), were integrated through epidemiology-guided feature engineering. To address class imbalance and temporal dynamics, Synthetic Minority Over-sampling Technique (SMOTE) resampling and sliding time-window features were applied. The proposed ensemble model combines Random Forest, XGBoost, and LightGBM using a soft-voting strategy, with logistic regression as a baseline. Results show that the ensemble model outperforms single models, achieving an AUC of 0.86 and a PR-AUC of 0.65. After threshold optimization, sensitivity increased from 0.78 to 0.87. Under field conditions, the system provided herd-level early warnings with an average lead time of approximately 12 days before confirmed outbreaks, demonstrating its feasibility and practical value for proactive brucellosis surveillance in high-altitude pastoral systems.

Applied Biosciences doi: 10.3390/applbiosci5020031

Authors: Dimitrios Morakis Athanasia Kotini Alexandra Giatromanolaki Adam Adamopoulos

Castrate-resistant prostate cancer (PCa) is a critical situation in which many patients will relapse. Hormonal androgen deprivation therapy (HADT) is the gold standard of care when a patient relapses, following primary surgical or radiation therapy. Usually, the benefits from HADT are poor and recurrent disease after HADT treatment is termed castrate-resistant prostate cancer (CRPC), which is in most cases fatal. The therapeutic regimens for CRPC include chemotherapy with docetaxel, immunotherapy agent sipuleucel-T, the taxane cabazitaxel, the CYP17 inhibitor abiraterone acetate and the androgen receptor (AR) antagonist enzalutamide. Thus, it is imperative to study the inherent property of prostate cancer cells, to resist therapy and reconsider the therapeutic protocols (continuous v’s intermittent). We make use of a hybrid mathematical model which consists of an extension of a very potent ordinary differential equation (ODE) Baez–Kuang model, combined with two Game Theory components: the Minority Game for adaptive behavior and the Axelrod model for heterogeneity behavior. Our study suggests that increasing tumor adaptability, through Minority Game dynamics, improves short-term prostatic-specific antigen (PSA) control and stabilizes therapy cycles. However, this comes at the cost of driving the tumor to a homogeneous, androgen-independent (AI) state, which is therapy-resistant. Conversely, maintaining heterogeneity, via Axelrod dynamics, sustains a mixed population, with androgen-dependent (AD) cells persisting longer and potentially delaying resistance emergence.

Applied Biosciences doi: 10.3390/applbiosci5020030

Authors: Génesis K. González-Quijano José Roberto González-Reyes Ilse Monroy-Rodríguez Esmeralda Rangel-Vargas Ciro Baruchs Muñoz-Llandes Fabiola Araceli Guzmán-Ortiz

Whey is a major by-product of the dairy industry and represents a valuable source of proteins that can be enzymatically converted into bioactive peptides with diverse health-related functions. In recent years, increasing attention has been given to whey-derived peptides due to their antioxidant, antihypertensive, antimicrobial, anti-inflammatory, antithrombotic, immunomodulatory, and anticancer activities, highlighting their potential use as functional ingredients and nutraceutical compounds. The generation and biological functionality of these peptides are strongly influenced by the protein source, processing conditions, enzymatic or microbial hydrolysis strategies, and peptide structure. Unlike the existing literature, this review provides an analysis of individual peptide sequences, meticulously linking their specific chemical structures to their diverse biological activities, such as antioxidants, antihypertensive, and immunomodulatory effects. By moving beyond general protein hydrolysis, this work offers a unique comparative framework that evaluates how these distinct peptide fractions perform under industrial conditions. Furthermore, it bridges the gap between laboratory discovery and commercial implementation, focusing on critical parameters for large-scale production, stability in functional food matrices, and the regulatory pathways required for market-ready nutraceuticals. This integrated approach provides a strategic roadmap for translating molecular bioactivity into high-value industrial applications. This review provides an applied overview of recent advances in the production of whey bioactive peptides, emphasizing enzymatic generation methods, structure–activity relationships, and underlying mechanisms of action associated with their biological effects. In addition, current and emerging applications of whey-derived peptides in functional foods, nutraceuticals, and health-oriented formulations are critically discussed. Finally, key challenges related to peptide stability, bioavailability, industrial scalability, and regulatory aspects are addressed to identify future perspectives for the effective translation of whey bioactive peptides from research to practical applications.

Applied Biosciences doi: 10.3390/applbiosci5020029

Authors: Theresa Bäuerle Christina Kunz Karlis Briviba

Phytate (myo-inositol hexakisphosphate) is a polyphosphate found in plant-based foods whose intestinal absorption mechanisms are insufficiently understood. Due to its high affinity for calcium, phytate can deplete extracellular calcium and potentially affect tight junction integrity, which could increase paracellular permeability. This study investigated the permeation of phytate across Caco-2 cell monolayers depending on calcium concentration. Differentiated Caco-2 cells were cultured on semi-permeable membranes and incubated with various phytate concentrations (0.17–1.66 mM) in media with low (2.1 µM) or normal (1.8 mM) calcium concentration. Phytate permeability and tight junction integrity were analyzed using HPLC and Lucifer yellow as a paracellular marker. At low calcium concentration, significant permeability was observed starting from 0.55 mM phytate (~60% at 1.66 mM), while at normal calcium concentration, significant permeability was only detectable at 1.66 mM. The increased Lucifer yellow permeation correlated with phytate permeation and confirmed tight junction disruption. Phytate that reached the basolateral side at physiological calcium concentration precipitated completely as insoluble calcium–phytate complex. These results demonstrate that phytate can pass intestinal epithelium via the paracellular pathway and increase the paracellular permeability, especially at low apical concentrations of calcium.

Applied Biosciences doi: 10.3390/applbiosci5020027

Authors: Vania Lizett Lucas-Hernández Liliana Lugo-Zarate Diana Patricia Olivo-Ramírez Estefani Yaquelin Hernández-Cruz José Pedraza-Chaverri Angélica Saraí Jiménez-Osorio

The food industry has a strong interest in lactic acid bacteria (LAB) because of their probiotic potential and health advantages. LAB have been previously isolated from pulque and agave sap, showing antibacterial action. However, their reaction to stress can limit their survivability, and their biological activities are strain-specific. To ascertain the impact of LAB isolated from pulque and agave sap on lifespan, thermal and oxidative stress, and health span parameters, we fed the nematode Caenorhabditis elegans these bacteria. The nematodes fed the Escherichia coli OP50 strain were utilized as a control for each experiment. Animals were fed each strain for four days starting from L4 and either (day 5) exposed to oxidative stress caused by high hydrogen peroxide concentrations (8 mM) or acute heat stress (35 °C) for four hours. The strains Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum significantly improved lifespan, fertility, movement, and heat shock resistance. Lacticaseibacillus casei enhanced the C. elegans lifespan, and Levilactobacillus brevis only increased its survivability in the heat shock studies. Interestingly, we discovered a harmful impact on animals fed Pediococcus acidilactici. This study highlights that, even when strains come from the same plant source, their biological activity might differ significantly.

Applied Biosciences doi: 10.3390/applbiosci5020028

Authors: Ivan T. Ivanov Boyana K. Paarvanova

Two spectrin-sensitive relaxations have been reported in the RBC plasma membrane: βs (1.4 MHz, related to the interface β-relaxation) and γ1s (9 MHz, rotation alignment of spectrin-bound dipoles by penetrating electric field). Here, a third (αs) relaxation type is reported within the frequency region of surface (α) relaxation. With low-ion-strength outside media, the adsorption of blood plasma immunoglobulins on RBCs was found to inhibit βs and γ1s relaxations, while αs relaxation was enforced with strong inflammation. The three relaxations are represented by three consecutive segments on the Cole′s plots: Δεrd″.ω against Δεr′ and Δεrd″/ω against Δεr′. Here, ω is the frequency of the field and Δεr* = Δεr′ + j.Δεrd″ is the change in the relative complex dielectric permittivity of RBC suspension at the denaturation temperature of spectrin. The βs segment in Δεrd″.ω against the Δεr′ plot could be regarded as a vector (complex number) whose projection on the vertical axis (the irreversible loss in energy) could express the ability of the plasma membrane to deform (under the impact of shear stress).

Applied Biosciences doi: 10.3390/applbiosci5020026

Authors: Beatriz Elena Castro-Valenzuela Tannia Janeth Vega-Montoya Blanca Sánchez-Ramírez Álvaro Vargas-Cázares Moisés Armides Franco-Molina M.Eduviges Burrola-Barraza

Bidirectional communication between the oocyte and surrounding follicular cells coordinates follicle growth, meiotic maturation, and the acquisition of competence. We aimed to identify genes related to follicular crosstalk and the secretory pathway as candidate mediators of cumulus–oocyte complex (COC) crosstalk in cattle. Expressed sequence tags (ESTs) from bovine COCs were retrieved from databases and screened for genes related to secretion and the secretory pathway using SignalP and SecretomeP, and transmembrane proteins were removed, yielding 13 candidate genes. Candidate expression was examined in two GEO RNA-seq datasets to assess enrichment in oocytes versus cumulus cells. RT–qPCR profiling across tissues and reproductive cell types enabled principal component analysis and correlation/network analysis, visualized as heatmaps and Cytoscape, revealing an INBHA-SERPINE2-SDF2L1 co-expression pattern. INHBA and SERPINE2 protein products are secreted, whereas SDF2L1 protein is a secretory pathway-associated, endoplasmic reticulum-resident chaperone. Promoter sequences of INHBA, SERPINE2, and SDF2L1 were scanned with FIMO using JASPAR motifs, identifying shared SMAD-associated motifs and FSH/cAMP-related motif families. The data support a co-regulation model in which endocrine FSH/cAMP and activin/TGF-β–SMAD inputs converge on a shared transcriptional program consistent with a putative INHBA–SERPINE2–SDF2L1 co-regulated module, linking cumulus extracellular matrix remodeling/protease control with oocyte ER protein folding capacity during COC maturation.

Applied Biosciences doi: 10.3390/applbiosci5020025

Authors: Sumeyye Altunok Mutlu Erdogan Aykut Ozkul

Background: Crimean-Congo hemorrhagic fever (CCHF) is a severe tick-borne viral disease with a high fatality rate, and no licensed vaccines are currently available. The nucleoprotein (NP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) plays a critical role in viral replication and immune recognition, making it a promising target for vaccine development. This study aimed to design and evaluate a multiepitope recombinant DNA vaccine targeting the NP of CCHFV. Methods: Cytotoxic T lymphocyte (CTL) epitopes from the NP were predicted via immunoinformatics approaches and systematically assessed for antigenicity, allergenicity, toxicity, hydrophobicity, and global population coverage. The selected epitopes were incorporated into four DNA vaccine constructs driven by a cytomegalovirus promoter, adjuvanted with human β-defensin 3 (hBD3), and fused to the reporter protein mRuby3. The constructs were evaluated in vitro using a fluorescent reporter system designed to provide a readout of TCR signaling upon the co-culture of T lymphocytes with differentiated monocytic cells expressing antigens. In vivo immunogenicity and protective efficacy were assessed in BALB/c (exploratory pilot) and IFNAR−/− mice, a highly susceptible model for viral infection. Cytokine responses were measured to assess immunogenicity. Results: In vitro assays showed predominantly antigen-independent T-cell activation, suggesting that nonspecific stimulation inherent to the reporter co-culture system likely obscured the detection of antigen-specific TCR signaling. In vivo analyses in BALB/c mice revealed that the constructs elicited only modest systemic cytokine profiles while CCHFV-specific IgG and IFN-γ secretion remained undetectable, indicating that antigen-specific T-cell and antibody responses were limited. In the IFNAR−/− challenge model, several peptide groups achieved significant 2–3 log reductions in tissue viral RNA and infectious titers (p < 0.05 vs. sham). However, the observed viral modulations were insufficient to reach the protective threshold and did not translate to a survival benefit (0%). Conclusion: Despite a rational in silico foundation, the multiepitope DNA vaccine constructs demonstrated limitations in inducing potent, antigen-specific immunity across both mouse models. The lack of antigen-specific responses indicates limitations in epitope selection, construct design, and delivery strategies, requiring optimization of next-generation epitope-based vaccines. These findings highlight the complexity of translating computational epitope predictions into functional vaccines, and provide benchmark data as a framework to guide future optimizations.

Applied Biosciences doi: 10.3390/applbiosci5020024

Authors: Jasmina Kurepa Jan A. Smalle

While “survival of the fittest” implies that competition is the main driver of evolution, cooperation and altruism are also widespread in nature, even among plants. This suggests that natural selection favors regulatory systems that balance competitive growth with restraint, depending on context. We propose that plant hormones are key mediators of this balance, acting along a spectrum from competition to cooperation. Based on evidence from developmental, ecological, and evolutionary studies, we classify major plant hormones by their roles in competitive behavior: auxin, gibberellins, and brassinosteroids drive competitive foraging and resource acquisition, while cytokinins, abscisic acid, strigolactones, ethylene, salicylic acid, and jasmonate are linked to growth restraint, resource conservation, and communal defense. This functional partitioning reflects a modular hormonal architecture that allows plants to adapt flexibly to their environment and social context. We explore how this classification could inform the use of plant hormones in agriculture and advance research in plant kin selection and inclusive fitness.

Applied Biosciences doi: 10.3390/applbiosci5020023

Authors: Latifa Bouissane Sohaib Khatib Reda El Boukhari Valérie Thiery Ahmed Fatimi

Cancer is an alarming health concern and economic burden in both developed and developing countries. Recently, there has been a growing demand for new alternative medications with more effectiveness and fewer harmful effects. During the past decades, a set of chemotherapeutic agents has been developed to fight against a large spectrum of cancer types. Unfortunately, their use is associated with a high level of toxicity; they are expensive, also, and their deployment is restricted by the emergence of cellular resistance. Plant-based components are garnering attention due to their low toxicity, selectivity, efficiency, and ease of accessibility. Alkaloids are one of these targeted compounds. Indeed, they are a highly diverse group with basic heterocyclic nitrogen-containing alkaloids that exhibit potent anticancer effects against a large panel of solid and liquid tumors, such as lung, breast, leukemia, liver, and colon cancer. The main molecular mechanisms involved in alkaloids’ anticancer effect are the induction of apoptosis via the extrinsic and intrinsic pathways, DNA damage, and the inhibition of cell cycle progression. Amazingly, these auspicious compounds exhibited strenuous inhibitory effects against a whole range of key enzymes involved in cancer progression and metastasis, such as Cytochrome P450 (CYP450), Cyclooxygenase-2 (Cox-2), Lysine-Specific Demethylase 1 (LSD1), Poly [ADP-ribose] polymerase (PARP), and topoisomerase, mainly through two action modes, namely irreversible and reversible inhibition. Furthermore, several conventional extraction methods have been developed to extract bioactive compounds from natural matrices, such as Soxhlet and hot water extraction. However, these techniques have many drawbacks, as they require a large amount of organic solvents, which not only affect human health but also generate severe environmental issues. To overcome these limitations, multiple eco-extraction techniques have emerged as potential alternatives to traditional extraction methods such as ultrasonic extraction, microwave-assisted extraction, and supercritical fluid extraction. In fact, they are considered eco-friendly and efficient technologies with less time and solvent consumption. Overall, this review aims to provide an updated overview of the most prominent anticancer alkaloids that have not been well reviewed already, as well as the main green extraction techniques relevant to the extraction of antineoplastic alkaloids.

Applied Biosciences doi: 10.3390/applbiosci5010022

Authors: Payal Ganguly

Musculoskeletal (MSK) diseases present major health and economic challenges globally. Advancing age, diseases like osteoarthritis (OA), osteoporosis (OP), fracture and other conditions significantly reduce the quality of life (QOL) of these patients. Current pharmaceutical approaches are able to manage symptoms for some of these; however, they do not provide long-term solutions. Surgeries which are usually the final resort, present an added layer of challenges with the risk of post-surgical complications. The last couple of decades have observed an increase in the use of tissue engineering and regenerative medicine (TERM) for bone tissue engineering (BTE) applications. With the advent of artificial intelligence (AI), there will inevitably be an intersection of AI with TERM for MSK conditions. As of 2025, AI is already in use for small-scale applications in BTE including data extraction, image analysis, scaffold design and fabrication using three-dimensional (3D) printing techniques. This review outlines the convergence of these three fields and discusses the potential of their intersection. The author describes the need for this convergence, a brief update of TERM in MSK in the last decade, followed by the potential of AI in MSK-TERM. The review concludes on the challenges and future directions of the emerging field and hopes to encourage bold and ambitious collaborations between industry, academia, hospitals and health-care start-ups to realize the potential of this unique intersection.

Applied Biosciences doi: 10.3390/applbiosci5010021

Authors: Ameer B. Alsultani Ameer R. Hassan Muntadher M. Hoom Halah I. Khani Katalin Kovacs Balazs Benyo Hussam Al-Saedi

This work presents a compact microwave sensor for noninvasive blood glucose monitoring based on a substrate-integrated waveguide loaded with a complementary split-ring resonator on RO4350. The sensing principle uses shifts in resonance frequency and changes in S-parameters to track the dielectric dispersion of glucose-containing tissue. The resonator is constructed using Substrate-Integrated Waveguide (SIW) technology, which mimics the propagation characteristics of a conventional rectangular waveguide. To validate its versatility, the sensor implements three practical sample delivery modes: direct liquid contact with the sensing surface, a glass tube holder mounted over the active region, and a non-invasive fingertip interface. Electromagnetic simulations and benchtop measurements confirm clear glucose-dependent frequency shifts with stable matching and insertion levels. Across the physiological range of 20 to 200 mg·dL−1, the sensor exhibits clear glucose-dependent resonance shifts in all configurations. In direct contact mode, the resonance frequency shifts from 10.83 GHz to 10.45 GHz with sensitivities up to 2.47 MHz per mg·dL−1. The tube configuration shows a shift from 10.49 GHz to 10.38 GHz with sensitivity up to 0.80 MHz per mg·dL−1, while reducing contamination. In the non-invasive fingertip mode, the resonance shifts from 2.56 GHz to 2.52 GHz with sensitivities up to 0.25 MHz per mg·dL−1. These results confirm the sensor’s compactness, reliability, and suitability for portable, low-cost glucose monitoring. The results indicate that the proposed sensor can support practical continuous or spot monitoring and offers a clear path toward portable and low-cost glucose assessment.

Applied Biosciences doi: 10.3390/applbiosci5010020

Authors: Luana Conte Angela Filoni Luca Schinzari Ester Sofia Congedo Lucia Pietroleonardo Rocco Rizzo Ugo De Giorgi Donato Cascio Giorgio De Nunzio Maurizio Congedo

Cutaneous melanoma is one of the most aggressive skin cancers, and early diagnosis remains essential to reduce mortality. Reflectance Confocal Microscopy (RCM) provides non-invasive, quasi-histological images of the epidermis, dermoepidermal junction (DEJ), and dermis, enabling real-time assessment of melanocytic lesions. However, interpretation still relies on expert visual evaluation, which is time-consuming and subjective. In this context, Artificial Intelligence (AI) and Computer-Assisted Detection (CAD) systems are emerging as valuable tools to improve diagnostic accuracy and reproducibility. This review summarizes research on AI applications in RCM imaging for melanoma, focusing on three major areas: delineation of skin strata, segmentation of tissues and morphological patterns, and classification of benign versus malignant lesions. Early approaches included Bayesian classifiers, wavelet-based decision trees, and logistic regression, while recent studies have employed support vector machines, random forests, and increasingly deep learning architectures such as convolutional and recurrent neural networks. The results demonstrate encouraging accuracy in DEJ localization, the segmentation of diagnostically relevant patterns, and the discrimination of melanoma from benign nevi. We distinguish the maturity of dermoscopy-based AI (AUC (ROC) > 0.80 on large multicenter cohorts) from the still-exploratory evidence for RCM-based AI. Nonetheless, current studies are often limited by small datasets, heterogeneous protocols, and a lack of multicenter validation. Overall, progress in AI applied to RCM supports the development of CAD systems that could assist clinicians during acquisition and diagnosis, reducing unnecessary biopsies and improving early melanoma detection. Future work should address standardization, dataset expansion, and the integration of advanced AI methods to move closer to clinical implementation.

Applied Biosciences doi: 10.3390/applbiosci5010019

Authors: Divya Jaroni Kaylee Rumbaugh

Zoonotic pathogens could persist in their environment and be introduced into the food-chain. With careful screening and selection, lactic acid bacteria (LAB) could be used as direct-fed microbials (DFMs) to control these pathogens in food animals. Previously isolated LAB (n = 250) were evaluated for inhibition against Shiga-toxigenic Escherichia coli (STEC) and Salmonella enterica, using agar spot test. Tests revealed that LAB were more effective against Salmonella than STEC, with 67% showing excellent (>15 mm) inhibition. LAB (n = 65) exhibiting significant pathogen inhibition (zones > 10 mm) were tested for acid (pH: 2, 4, 5, 7) and bile (0, 0.1, 0.3, 0.5%) tolerance, and biofilm-forming capabilities. About half of the tested LAB exhibited excellent to very good tolerance. All LAB formed biofilms, with 33% forming strong biofilms. LAB (n = 59) were also examined for susceptibility to commonly used antibiotics due to their intrinsic or acquired antibiotic resistance (AR), transferrable to pathogens. Only S. thermophilus S-2 showed susceptibility to all the antibiotics. The majority were susceptible to erythromycin (88%), followed by ampicillin (85%), clindamycin (64%), tetracycline (58%), vancomycin (44%), streptomycin (15%), and gentamicin (9%). Overall, LAB showed strong inhibition against pathogens, along with survival capabilities for environmental stress conditions, and could be considered for potential DFM applications.

Applied Biosciences doi: 10.3390/applbiosci5010018

Authors: Andres Zapata Betancur Freddy Forero Longas Adriana Pulido Diaz

Growing interest in natural therapies has increased the demand for essential oils; however, the complex interactions within their mixtures that dictate their final efficacy remain poorly understood. This study aimed to optimize a blend of ginger, cinnamon, tea tree, and geranium essential oils to develop an active ingredient, with synergistic multifunctional bioactivities, that was relevant to cutaneous healing. Initially, the composition and cytotoxicity for individual oils were determined; subsequently, a D-optimal mixture design was employed to evaluate three biological responses related to skin recovery: ultraviolet B radiation absorption, red blood cell lysis inhibition, and catalase enzyme activity. GC-FID analysis revealed the following major components (% w/w): cinnamon (cinnamaldehyde, 77.56%), ginger (α-zingiberene, 33.77%), geranium (citronellol, 33.6%), and tea tree (terpinen-4-ol, 38.38%). Dose–response data from essential oils tested against Detroit ATCC 551 skin fibroblasts revealed a clear cytotoxic hierarchy (IC50 µg/mL): cinnamon (21.03) > ginger (25.3) > tea tree (41.67) > geranium (92.51). Cinnamaldehyde content was the primary contributor to photoprotective capacity, with a maximum sun protection factor (SPF) of 4.5. Inhibition against erythrocyte membrane lysis was not attributable to a single component; maximum protection (98.4%) was achieved through synergy between oxygenated monoterpenoids (geranium and tea tree), sesquiterpenes (ginger), and aromatic aldehydes (cinnamon). Highest catalase activity (160.86 kU/g Hb) was reached in mixtures with high cinnamaldehyde and eugenol contents, whereas an antagonistic effect was observed between tea tree and geranium oils. Finally, an optimal formulation (desirability = 0.927) was identified (% w/w): 31.7% ginger, 39.1% cinnamon, 14.5% tea tree, and 14.7% geranium. Experimental validation confirmed no significant difference compared with developed predictive models. This optimized mixture constitutes a bioactive natural component with potential for use in products aimed at promoting skin health, warranting further investigation into direct models of skin healing.

Applied Biosciences doi: 10.3390/applbiosci5010017

Authors: Doha A. Mohamed Hagar F. Elbakry Gamil E. Ibrahim Sherein S. Abdelgayed Hoda B. Mabrok

Hyperlipidemia is a major global risk factor for cardiovascular disease, underscoring the need for safe, multi-target preventive strategies. In this study, two novel dietary supplements were developed by blending freeze-dried aqueous extracts of chamomile (CDS) or thyme (TDS) with linseed oil (1:1, w/w) and evaluated for their phytochemical composition, antioxidant capacity, and hypolipidemic efficacy. Total phenolics, total flavonoids, fatty acid composition, volatile constituents, and individual phenolic profiles were characterized, while antioxidant activity was assessed using DPPH· radical-scavenging and FRAP assays. Hypolipidemic activity was investigated in a Triton X-100-induced hyperlipidemia rat model through an assessment of plasma lipid parameters, oxidative stress and inflammatory markers, and liver and kidney function indices, supported by hepatic histopathology. Molecular docking was performed to explore the interactions of major bioactive compounds with AMP-activated protein kinase (AMPK) and HMG-CoA reductase. Both CDS and TDS exhibited strong antioxidant activity and high polyphenol content, with kaempferol and chlorogenic acid identified as the predominant phenolics in CDS and TDS, respectively. β-Farnesene and carvacrol were the main volatile constituents. In vivo, both formulations significantly reduced total cholesterol, triglycerides, LDL-C, lipid peroxidation markers, and TNF-α, while increasing HDL-C and improving cardiac risk indices, with more pronounced effects observed for TDS. Histopathological analyses confirmed marked hepatoprotection, particularly in the TDS-treated group. Docking analyses identified ellagic acid as the strongest dual binder to both AMPK and HMG-CoA reductase. Overall, these findings demonstrate that chamomile-linseed and thyme-linseed formulations exert synergistic, multi-target antioxidant and hypolipidemic effects, supporting their potential as nutraceutical strategies for the early prevention and management of hyperlipidemia and cardiometabolic risk.

Applied Biosciences doi: 10.3390/applbiosci5010016

Authors: Rumyana Eneva Stephan Engibarov Irina Lazarkevich Yana Gocheva Ekaterina Krumova Radoslav Abrashev Jeny Miteva-Staleva Vladislava Dishliyska Lyudmila Yovchevska Katerina Zaharieva Ognian Dimitrov Virginia Spasova Ivan Kolev Milen Dimov Irina Stambolova

Nanotechnology has emerged as a promising option in combating the worsening situation with antibiotic resistance. We studied the antimicrobial effectiveness of four types of green synthesized zinc oxide nanoparticles (ZnO-NPs), obtained via Pluronic-assisted co-precipitation by lavender and thyme essential oils and their praseodymium-doped variants. Resazurin Microtiter Assay was applied to a panel of Gram-positive and Gram-negative bacteria from Risk 1 and 2 groups and the ESKAPE group. In relation to the pro-oxidative features of the ZnO-NPs, the production of superoxide dismutase (SOD) and catalase (CAT) in the tested microorganisms was also investigated, as these enzymes are important participants in the antioxidant defense of the bacterial cell and are considered virulence factors. We hypothesized that the sensitivity of microorganisms to the action of ZnO-NPs is related to their innate levels of antioxidant enzyme activity. The results showed that all types of studied ZnO-NPs had an antibacterial effect against the entire panel of tested strains, but with different potencies. The strongest effect was found for Arthrobacter nicotianae, Oerskovia paurometabola, Bacillus subtilis, and Escherichia coli. Less inhibition was observed for bacteria from Risk group 2 maybe due to their better antioxidant protection, especially for Pseudomonas aeruginosa. Praseodymium doping contributed to enhancing the bactericidal effect. A correlation between susceptibility of bacteria to ZnO-NPs and their antioxidant enzyme activity was observed.

Applied Biosciences doi: 10.3390/applbiosci5010015

Authors: Gulsun Akdemir Evrendilek

Seafood products are highly perishable and particularly susceptible to contamination by pathogenic and spoilage microorganisms, including Listeria monocytogenes, Vibrio spp., Salmonella spp., and Escherichia coli. Conventional control strategies in seafood processing and storage largely rely on chemical preservatives and thermal treatments, which may negatively affect sensory quality and increasingly conflict with consumer demand for minimally processed, “clean-label” foods. In this context, bacteriophages, viruses that specifically infect and lyse bacterial hosts, have emerged as natural, targeted, and environmentally sustainable biocontrol agents for food safety applications. This review provides a comprehensive assessment of bacteriophage applications in seafood processing and storage, with particular emphasis on their mechanisms of action, host specificity, and ability to selectively reduce pathogenic bacteria without compromising nutritional or sensory attributes. Recent advances in phage-based technologies, including phage cocktails, immobilized phage systems, and genetically engineered phages, are discussed in relation to their efficacy against major seafood-associated pathogens under both laboratory and industrial conditions. Key challenges limiting large-scale implementation such as phage resistance development, regulatory considerations, stability during processing and storage, and consumer perception are critically evaluated. In addition, the review highlights emerging evidence on the synergistic use of bacteriophages with complementary preservation strategies, including natural antimicrobials and innovative packaging systems. Overall, this review underscores the potential of bacteriophage-based interventions as practical and sustainable tools to enhance seafood safety, extend shelf life, and support modern seafood processing practices aligned with evolving regulatory and consumer expectations.

Applied Biosciences doi: 10.3390/applbiosci5010014

Authors: Daniela Hristov Done Stojanov

Complex transcriptional and epigenetic regulation, including variation in promoter-level cis-regulatory architecture, influences infertility. In this study, we performed a purely in silico analysis of the −1000 to −1 bp promoter regions (relative to the annotated TSS) of 13 human fertility-related genes using an integrated motif-discovery and annotation workflow (NNPP, MEME/STREME, Tomtom, FIMO/CentriMo, GOMo, and MethPrimer). Motif discovery identified multiple statistically supported de novo promoter motifs, and motif scanning mapped their occurrences across the analyzed promoters. Similarity searches against curated PWM databases did not yield significant matches under stringent criteria, consistent with divergent or under-represented motif patterns. Functional association analysis and CpG island profiling further highlighted promoter segments that merit prioritization for follow-up testing. As the analysis is purely in silico and restricted to a fixed promoter window, the identified motifs should be interpreted as candidate regulatory elements pending experimental validation.

Applied Biosciences doi: 10.3390/applbiosci5010013

Authors: Olabisi Tajudeen Obafemi Ademola Olabode Ayeleso Blessing Ariyo Obafemi Jeremiah Oshiomame Unuofin Adewale Odunayo Oladipo Sogolo Lucky Lebelo Monde Ntwasa

The present study aims to evaluate the interaction of gliclazide with proteins related to inflammation—{inhibitor of nuclear factor kappa-B kinase subunit beta (IKKα) and NF-kappa-B-inducing kinase (NIK)}; oxidative stress—{kelch domain of Kelch-like ECH-associated protein 1 (KKeap1)} and ER stress—{inositol-requiring enzyme-1alpha (IRE1α)}. X-ray crystal structure of IKKα, (PDB ID: 5EBZ), KKeap1 (PDB ID: 4L7B), NIK (PDB ID: 8YHW) and IRE1α (PDB ID: 4YZ9) were obtained from Protein Data Bank and Open Babel 3.1.1 was used to prepare the ligands. Prior to docking, protein structures were prepared by removing water molecules, adding hydrogen atoms, and optimizing side chain conformations using Maestro (Schrödinger Suite, version 2024-2) along with the OPLS4 force field. Ligand docking was performed using the Glide application. Molecular dynamics simulation was performed with Desmond (Schrödinger Suite) within the Maestro interface for 100 ns for the NPT ensemble at 300 K and 1 atm pressure. Physicochemical and pharmacokinetics properties were analyzed using ADMETlab 3.0 and SwissADME. The binding energies of gliclazide with IKKα, NIK, KKeap1 and IRE1α were −8.3, −7.9, −8.4 and −8.8, respectively. Root mean square displacement (RMSD), root mean square fluctuation (RMSF) and radius of gyration analyses predicted relatively strong and stable interactions between gliclazide and the proteins, with favourable pharmacokinetic properties. It was also observed that CYP3A4 metabolizes gliclazide, in addition to CYP2C9 and CYP2C19. The activity of gliclazide against inflammation, oxidative stress and endoplasmic reticulum stress might be via interaction with these proteins.

Applied Biosciences doi: 10.3390/applbiosci5010012

Authors: Serban Iancu Papacocea Ioana Anca Bădărău Toma Marius Papacocea

Despite significant advances in neurosurgical and critical care, traumatic brain injury (TBI) remains a major cause of morbidity and mortality. Surgical treatment of intracranial hemorrhagic lesions can only target the primary mechanical injuries and their immediate consequences but fails to address the biochemical pathological cascade that unfolds during the second injury. This review synthesizes current knowledge regarding the use of several biomarkers in diagnosis and prognosis assessment. A structured literature search was conducted by querying the PubMed database. Articles evaluating diagnostic and prognostic biomarkers in adult TBI were screened according to Prisma guidelines, and data regarding biomarkers type, cut-off values, and correlations with the outcome were extracted and summarized. Among Central Nervous System (CNS)-Specific markers, S100 calcium-binding protein (S100B) emerged as a remarkably strong negative predictor for Computed Tomography (CT)-visible intracranial lesions (NPV = 97.3–100%), whereas glial fibrillary acidic protein (GFAP) yielded both high NPV and brain specificity. Coagulation parameters such as the international normalized ratio (INR) and fibrinogen were independently correlated with mortality and unfavorable outcomes. Fibrinogen displayed a bidirectional relationship with increased mortality risk at both low (<2 g/L) and high (>4.5 g/L) values. In conclusion, biomarkers quantify the otherwise invisible progression of secondary traumatic brain injury that persists even after successful surgery.

Applied Biosciences doi: 10.3390/applbiosci5010011

Authors: Abdmajid Saad Hwej Mohammed Alsharif Ali Al-Ferjani Simon Kennedy

Background: Caveolin-1 (Cav-1) is a protein found in various forms and locations within cells and tissues throughout the body. Studying its structure and function provides valuable insights into key cellular processes such as growth, death, and cell signaling. This review synthesizes evidence from human studies and animal models to elucidate the complex role of Caveolin-1 (Cav-1) in regulating nitric oxide (NO) synthesis within the vasculature and perivascular adipose tissue (PVAT) during atherosclerosis. Cav-1 is a master regulator of endothelial NO synthase (eNOS), a relationship well-defined in rodent endothelial cells and cell lines. In humans, loss-of-function CAV1 mutations are linked to pulmonary arterial hypertension, suggesting a protective vascular role. Paradoxically, Cav-1 is upregulated in atherosclerotic plaques. Whether this represents a pathological process reducing NO bioavailability or a compensatory response remains unclear. Furthermore, the direct translation of the Cav-1/eNOS axis to PVAT—a metabolically active tissue expressing Cav-1—is not fully established outside of preclinical models. PVAT influences vascular tone and inflammation, potentially contributing to the paradoxical, stage-specific roles of Cav-1 in disease. Resolving these questions requires integrating human observational data with mechanistic insights from animal models to evaluate Cav-1 as a therapeutic target in vascular disease.

Applied Biosciences doi: 10.3390/applbiosci5010010

Authors: Yumna Ladha Sushmita Sanaka Adam Burke Royston Goodacre Karina T. Wright Jade Perry Charlotte H. Hulme

Metabolic profiling enables comprehensive characterisation of the small molecules that are part of the biochemical composition of biological fluids. The most widely profiled biofluids include serum and plasma. Additionally synovial fluid provides a direct reflection of the metabolomic environment of joints and holds promise for biomarker discovery in arthropathies. However, the reproducibility of metabolomics data is highly sensitive to pre-analytical variation, and at the present time, standardised protocols for synovial fluid remain underdeveloped. This review aims to identify and evaluate the existing literature on effects of biofluid pre-analytical handling treatments on metabolic profiles. This review was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines. A search was carried out to identify studies employing LC-MS, GC-MS, and NMR spectroscopy for the investigation of factors including sample collection variables, pre-centrifugation conditions, centrifugation parameters, post-centrifugations conditions, sample storage conditions, and freeze/thaw cycling. Best practice recommendations emerging from this review include the use of additive free serum and heparin plasma tubes, the centrifugation of samples within two hours of collection, immediate storage of samples at −80 °C, and avoidance of repeated freeze/thaw cycling. However, while pre-analytical influences have been extensively characterised for plasma and serum, evidence for synovial fluid remains limited. Overall, the findings highlight the existing recommendations for plasma and serum and demonstrate the need for standardised pre-analytical protocols and validation of quality control markers to advance synovial fluid metabolomics.

Applied Biosciences doi: 10.3390/applbiosci5010009

Authors: Olivia Torres-Bugarín Isaac Gómez-Ferreyra Mario Contreras-Dueñas María Luisa Ramos-Ibarra Rafael Bello-Bedoy Alejandro Sánchez-González Francisco Casillas-Figueroa Balam Ruiz-Ruiz Karina Iveth Orozco-Jiménez María Evarista Arellano-García

Background: The Allium cepa test is a widely used, cost-effective, and versatile model for assessing cytogenotoxicity. Cytotoxicity is determined through changes in root growth and the mitotic index, while genotoxicity is identified through chromosomal aberrations such as breaks, bridges, and micronuclei. Objective: To synthesize the methodological principles, applications, and interpretation of the assay’s endpoints, with emphasis on environmental monitoring, nanotoxicology, and the evaluation of emerging materials. Methods: An exploratory analytical approach was applied to identify and compare studies employing the Allium cepa assay across different contexts. The literature, selected from scientific databases, was organized to highlight methodological diversity and biomarker performance. Conclusions: Compared with other models, Allium cepa stands out for its simplicity, the availability of multiple cytogenotoxic markers, and its minimal ethical constraints, making it especially suitable for research in low-infrastructure settings. Future studies should work toward the international standardization of methodologies, the integration of this model with molecular and omics-based approaches, and its incorporation into predictive frameworks for environmental and human health risk assessment. In an increasingly complex toxicological landscape, Allium cepa emerges as a pivotal tool for enhancing toxicological surveillance and safeguarding biological systems.

Applied Biosciences doi: 10.3390/applbiosci5010008

Authors: Maximiliano Sandoval Dylan Zapata Dácil Rivera Elkin Y. Suarez-Villota Boris Parra

Escherichia coli is a major pathogen responsible for urinary tract infections, septicemia, and other clinically relevant conditions, with increasing multidrug resistance (MDR) limiting available treatment options. In this context, bacteriophages represent a valuable resource for exploring novel antimicrobial and biotechnological tools. Here, we report the isolation and genomic characterization of BorMax, a novel lytic phage infecting multiple MDR E. coli. Transmission electron microscopy revealed a tailed morphology consistent with Dhillonvirus. Whole genome sequencing and de novo assembly showed a linear double-stranded DNA genome of 45,502 bp, encoding 70 predicted coding sequences (CDSs) and lacking tRNAs. Bioinformatic analyses confirmed the absence of lysogeny-associated genes, as well as virulence and antimicrobial resistance determinants. Comparative genomics using classified BorMax within the genus Dhillonvirus as a new species, sharing <77% intergenomic similarity with known members. Notably, predictions using PaCRISPR and AcRanker identified four CDSs with strong anti-CRISPR (Acr) potential, representing previously undescribed Acr candidates in this group. These genomic features highlight the novelty, safety, and potential biotechnological relevance of BorMax and contribute to the expanding genomic and functional diversity of Dhillonvirus and E. coli-infecting phages.

Applied Biosciences doi: 10.3390/applbiosci5010007

Authors: Giuseppa D’Amico Melania Ionelia Gratie Giuseppe Vergilio Francesco Cappello Alessandra Maria Vitale

The heat shock protein 60 (Hsp60) is a highly conserved molecular chaperonin belonging to the chaperone system, a complex network that maintains proteostasis and regulates numerous cellular processes beyond protein folding. Initially described as a mitochondrial protein essential for the folding of newly imported polypeptides, Hsp60 is now recognized as a multifunctional molecule. Its expression, localization, and post-translational modifications dynamically influence cell fate and tissue homeostasis. Alterations in Hsp60 quantity, structure, or distribution underlie a heterogeneous group of disorders known as chaperonopathies, which may occur “by defect,” “by excess,” or “by mistake” (also called “by collaborationism”). Genetic Hsp60’s chaperonopathies are associated with rare neurodegenerative and cardiovascular diseases, whereas acquired forms contribute to widespread conditions, including autoimmune, inflammatory, and malignant pathologies. This review provides a comprehensive overview of Hsp60 biology across human systems, emphasizing its structural plasticity, context-dependent functions, and dual role in health as both a biomarker and a therapeutic target. The emerging paradigm of chaperonotherapy, encompassing positive strategies to restore protective chaperones and negative strategies to inhibit pathogenic ones, highlights the translational potential of targeting Hsp60. Understanding the molecular mechanisms governing its activity will be essential for developing precision medicine approaches aimed at modulating the chaperone system in human disease.

Applied Biosciences doi: 10.3390/applbiosci5010006

Authors: Laverdure Tchamani Piame Yandiswa Yolanda Yako

Probiotics, whether consisting of a single strain or multiple strains, are attracting growing interest in the management of type 2 diabetes mellitus (T2DM). However, their efficacy remains a matter of controversy and requires careful consideration. Accordingly, this meta-analysis aimed to compare the efficacy of single-strain to that of multi-strain probiotics supplementation on glycated haemoglobin (HbA1c) and fasting blood glucose (FBG) levels in adults with T2DM. Nineteen articles published between 2017 and 2024 obtained from 4 databases (Cochrane, Web of Science, Scopus, and PubMed) were included. These interventions, conducted in a total of 1159 participants, lasted from 6 to 24 weeks and were based on clearly identified probiotic formulations, with assessments of HbA1c and FBG. The results showed that, overall, probiotic supplementation had no significant effect on HbA1c (−0.24%; 95% CI [−0.76; 0.27]; p = 0.36), although a trend towards reduction was observed for single-strain formulations (−0.57%; p = 0.05). Regarding FBG, only the multi-strain group showed a significant reduction (−0.76; 95% CI [−1.18; −0.34]; p < 0.001), while the effect of the single-strain formulation was not significant. The comparison between the two formulations (Wald test) showed that there was no significant difference (p ≤ 0.05). However, high heterogeneity (I2 > 75%) and variable strains/doses limit confidence in these findings.

Applied Biosciences doi: 10.3390/applbiosci5010005

Authors: Nattawut Chumnoi Papinwich Paimsang Watcharaporn Cholamjiak Tipwan Suppasat

This study aimed to develop a robust morphometric-based framework for classifying Apis cerana populations using deep learning and machine learning approaches. Previous studies on Apis cerana population differentiation have primarily relied on manual morphometrics or genetic markers, which are labor-intensive and often lack scalability for large image-based datasets. Forewing landmarks were automatically detected through a deep learning model employing a heatmap regression and Hourglass Network architecture. The extracted coordinates were processed by Principal Component Analysis (PCA) for dimensionality reduction, and shape alignment was further refined through Procrustes ANOVA to minimize non-biological variation. Nine machine learning algorithms were trained and compared under identical preprocessing and validation settings. Among them, the Extra Trees classifier achieved the highest accuracy (99.7%) in distinguishing the three populations—A. cerana cerana from China and A. cerana indica from Thailand, the northern and southern populations. After applying error-based data filtering and retraining, classification accuracy improved further, with almost perfect population separation. The Procrustes ANOVA confirmed that individual variation significantly exceeded residual error (Pillai’s trace = 1.13, p < 0.0001), validating the biological basis of shape differences. Mahalanobis distance and permutation tests (10,000 rounds) revealed significant morphological divergence among populations (p < 0.0001). The integration of geometric alignment and ensemble learning demonstrated a highly reliable strategy for population identification, supporting morphometric and evolutionary studies in Apis cerana.

Applied Biosciences doi: 10.3390/applbiosci5010004

Authors: Sumarin Soonsanga Boonhiang Promdonkoy

Mtx1 is a mosquitocidal protein that exhibits high toxicity toward Culex species. It is produced during the vegetative phase of Lysinibacillus sphaericus but at very low levels and is rapidly degraded. The low expression appears to result from a weak promoter and a potential regulatory stem-loop structure in the 5′ untranslated region. To investigate this regulation, promoter variants of mtx1 were constructed to disrupt stem-loop formation, and promoter activity was assessed using green fluorescent protein (GFP) as a reporter. Disruption of the inverted repeat resulted in approximately twofold higher fluorescence compared with the wild-type promoter in L. sphaericus 2297, indicating partial derepression of translation. To improve protein stability, Bacillus subtilis WB800N, a protease-deficient host, was employed for heterologous expression. Truncated Mtx1 (tMtx1) was secreted into the culture medium, and no obvious degradation products were detected by Western blot analysis under the conditions tested. Although the overall yield was low and not quantitatively determined, the secreted protein retained biological activity. Larvicidal assays showed elevated mortality in tMtx1-containing culture supernatants, with an estimated LC50 at approximately a 1:83 dilution and detectable activity up to a 1:512 dilution relative to control cultures. These results demonstrate that the upstream inverted repeat contributes to partial repression of mtx1 expression in L. sphaericus and that protease-deficient B. subtilis can be used as a host for producing biologically active tMtx1, although further optimization will be required to improve yield.

Applied Biosciences doi: 10.3390/applbiosci5010003

Authors: Alexander Ermoshin Marina Byzova Chaomei Ma Irina Kiseleva

One of the global challenges is the deficit of food. Food production is highly dependent on the productivity of agricultural plants used by humans and livestock. Various chemical and natural compounds are used to stimulate plant growth and increase their resistance to stress. The aim of our study was to analyze the chemical composition of extracts of the most common Ural tinder fungi and their effect on the early stages of wheat growth. Water–alcohol extracts from five wood-destroying fungi contained biologically active compounds (BACs), such as phenolics, free amino acids and reducing sugars. F. pinicola was characterized by the smallest amount of extracted substances. F. fomentarius has the largest amount of phenolic compounds and sugars, and I. obliquus had the highest concentration of free amino acids. Qualitative analysis revealed alkaloids in P. betulinus, and anthraquinones in F. fomentarius. Saponins were found in all tested species, except F. fomentarius. The extracts stimulated the early stages of wheat development at concentrations of 1.0–0.2 g of fungal biomass per liter. Seed germination rate was comparable to the control samples or exceeded it, and the length of roots and shoots increased. Thus, extracts from fruiting bodies of studied fungi can be recommended for priming wheat seeds, and for biotechnological cultivation.

Applied Biosciences doi: 10.3390/applbiosci5010002

Authors: Miguel Salvador-Adriano Miguel Angel Reyes-López José Alberto Narváez-Zapata Raymundo Rosas-Quijano Didiana Gálvez-López

Fruit softening in Carica papaya L. is a significant postharvest limitation, primarily driven by the dynamic remodeling of cell wall polysaccharides. In this study, we conducted a genome-wide identification and in silico characterization of gene families involved in cell wall assembly and disassembly in papaya. A total of 181 genes were identified and classified into metabolic pathways: hemicellulose (58), pectin (69), extensin (24), expansin (13), and cellulose (17). These genes encode 176 predicted proteins, ranging in size from 100 to 1093 amino acids, featuring family-specific catalytic domains, including glycosyl hydrolases, transferases, and serine/threonine kinases. Phylogenetic analyses revealed strong conservation within the expansin-A and pectin polygalacturonase subfamilies, while hemicellulose-related XTH genes exhibited significant diversification. Experimental validation of nine XTH members confirmed this diversification, with amplicons ranging from 322 to 1370 bp, consistent with computational predictions. Notably, CpXTH1 and CpXTH32 produced bands of approximately 1200 and 1400 bp, respectively. These findings underscore the complexity of papaya cell wall gene families and provide a molecular framework for understanding fruit softening. Given that postharvest losses of papaya in Mexico exceed 34.7% of production (approximately 150,000 tons annually), our results offer valuable genomic resources for biotechnological strategies aimed at extending shelf life and reducing economic losses.

Applied Biosciences doi: 10.3390/applbiosci5010001

Authors: Anastasia Ryapolova Daniil Shevyrev Angela Tsvetkova Olesya Sokolova Olga Vasileva Vladimir Andriianov Bogdan Krapivin Alena Kozlova Kristina Vorona Sofia Kondratieva Roman A. Ivanov Alexander Karabelsky Vasiliy Reshetnikov Ekaterina Minskaia

The search for effective anti-cancer therapies is one of the most significant goals of modern medicine. The combination of oncolytic viruses (OV) and mRNA immunoadjuvants can significantly improve the outcome or even substitute traditional immunotherapy. In addition to the direct OV-mediated cytotoxic elimination of tumor cells, both OV and mRNA immunoadjuvants can significantly alter the immunosuppressive tumor microenvironment (TME) supporting cancer cells and unleash the immune response against the malignant cells. The present study is aimed at assessing the therapeutic effects of recombinant vesicular stomatitis virus (rVSV) and lipid nanoparticles (LNP) delivering mRNA coding for murine interleukin-12 (mIL12) and granulocyte-macrophage colony-stimulating factor (mGMCSF) (mRNA-LNP) in colorectal carcinoma CT26-induced tumors both as independent therapies and in combination with each other. The results of the in vivo experiment on BALB/c mice demonstrated that rVSV monotherapy did not have a significant effect, with the tumor growth inhibition index (TGII) ranging from 13.7 to 29.8% on days 6–10 after the therapy start. While monotherapy with mRNA-LNP was more effective (TGII of 48.6–53.7%), it was the therapy combining the two approaches (rVSV and mRNA-LNP) that resulted in the highest TGII of 66.7% on day 10. While these results can be further improved by optimizing the experimental design, they show the great potential of combination immunotherapy for the treatment of oncological diseases.

Applied Biosciences doi: 10.3390/applbiosci4040058

Authors: Yana Gocheva Lyudmila Dimitrova Maya Zaharieva Venelin Hubenov Yana Ilieva Petya Orozova Vyacheslav Ilyin Denis Korshunov Ivan Simeonov Plamen Angelov Hristo Najdenski

Urban areas and suburbs are facing many environmental problems, one of which is the increasing pollution due to the accumulation of cellulosic waste. This article presents a laboratory study on the microbial biodegradation processes of paper as a cellulosic substrate under anaerobic and mesophilic cultivation conditions. A bacterial consortium with cellulose-degrading activity, as well as 4 individual strains originating from a methanogenic anaerobic bioreactor (BRA), was isolated, identified, and characterized. The results demonstrated that the consortium degraded 57.14% of the cellulose matrix within 20 days. Among the individual colonies, colonies 1 and 2 (identified as Clostridium tertium and Agromyces rhizospherae) exhibited lower activities (35.37% and 34.79%, respectively), while colony 3 (Clostridium paraputrificum) displayed the highest activity (83.74%). The mixture of all four colonies achieved lower degradation (21.22%). The performed metagenomic analysis of the microbial consortium revealed a wide variety of different bacterial genera, among which Clostridium, Bacteroides, and Ruminiclostridium dominate, and the species Bacteroides oleiciplenus, Clostridium butyricum, and Ruminiclostridium papyrosolvens. Scanning electron microscopy visualized the adhesion and morphological features of the degrading microbial population. Additional experiments on the development of a laboratory model for the anaerobic biodegradation of cellulose were carried out in BRAs by using different working volumes. A maximal level of cellulose decomposition was achieved in the BRA with a working volume of 1 L, reaching 71.0% cellulose decomposition on day 20. Long-term storage studies confirmed the survival and well-preserved activity of the consortium and individual isolates, demonstrating their potential for the development of bioconversion technologies.

Applied Biosciences doi: 10.3390/applbiosci4040057

Authors: Māris Seņkovs Laila Dubova Ina Alsiņa Ingrīda Krīgere Dagnija Lazdiņa Viktorija Vendiņa Sandra Marcinkeviča Vizma Nikolajeva

The conversion of livestock manure and peat into value-added fertilizers provides an environmentally sustainable approach to nutrient recycling and waste management. In this study, organic fertilizers were formulated from poultry, pig, and cattle manure mixed with peat and wood ash, with or without inoculation of the phosphate-solubilizing bacterium Priestia megaterium. Their efficiency was evaluated through plant growth and soil microbiological experiments involving conifer seedlings, herbaceous crops, and ornamental plants. Germination and growth trials with Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) revealed clear species-specific responses: spruce seedlings performed best in substrates containing poultry or cattle manure, while pine showed enhanced growth with pig manure combined with bacterial inoculant. In pansies (Viola × wittrockiana), growth responses varied by cultivar; cattle manure enriched with bacteria increased leaf projection area, whereas poultry manure markedly suppressed growth. For cucumbers, basil, barley, radish, and garden beans, yields were lower than with mineral fertilizers, yet bacterial inoculation significantly influenced soil microbial activity by modifying respiration rates and hydrolytic enzyme intensity in plant- and manure-specific ways. The results demonstrate that microbial supplementation can alter soil biological processes and nutrient turnover, though its effects on plant productivity remain inconsistent. Further research is required to assess long-term performance under field conditions, as practical application will depend on achieving stable and reproducible results.