BioTech doi: 10.3390/biotech15020045

Authors: Fidel Delgado Mario Blanco-Vieites María Álvarez-Gil Víctor Casado-Bañares Eduardo Rodríguez

Phycocyanin, a phycobiliprotein derived from the cyanobacterium Limnospira (Arthrospira) platensis (commonly known as Spirulina), is recognized for its antioxidant, immunomodulatory, and bioactive properties. This research aims to develop a new cosmetic ingredient based on phycocyanin incorporated into a high-lipid matrix, such as shea butter. A comprehensive characterization of the cytotoxicity and anti-inflammatory activity of this new bioactive phycocyanin–protein complex in human THP-1 monocytic cells was performed. For this purpose, cytocompatibility was evaluated using MTT assays at concentrations ranging from 10 to 0.0006% v/v. Anti-inflammatory activity was measured under LPS-induced inflammatory stress by measuring IL-6 and IL-8 secretion using ELISA in PMA-differentiated THP-1 cells treated with non-cytotoxic concentrations (0.04, 0.02, and 0.01% v/v). A crucial finding was the absence of anti-inflammatory activity at 0.01% v/v, indicating a minimum effective concentration threshold and, consequently, effective doses. The results of this research indicate that the phycocyanin and shea butter ingredients demonstrate strong cytocompatibility at relevant cosmetic doses and significant anti-inflammatory activity, supporting their suitability for formulations targeting skin sensitivity, erythema reduction, and post-inflammatory recovery.

BioTech doi: 10.3390/biotech15020044

Authors: Yota Yamada Mana Nemoto Motoyasu Miyazaki Hitomi Hirata Konatsu Hosogoe Akio Nakashima Hisako Kushima Hiroshi Ishii Osamu Imakyure

Clostridioides difficile infection (CDI) remains a major healthcare-associated infection, and metronidazole (MNZ) is still used for selected patients with non-severe infection in Japan. Although Clostridium butyricum MIYAIRI 588 (CBM588) may be used as an adjunctive therapy, clinical evidence regarding its add-on value to MNZ in non-severe CDI remains limited. We conducted an exploratory single-center retrospective cohort study of adults with non-severe CDI treated with MNZ between April 2015 and March 2025. Of the 161 patients diagnosed with C. difficile infection, 53 patients met the eligibility criteria and were analyzed. Patients were categorized into MNZ monotherapy (n = 27) or MNZ plus CBM588 combination therapy groups (n = 26), and clinical outcomes were compared. Clinical cure was achieved in 51.9% of patients in the monotherapy group and 69.2% of patients in the combination therapy group; however, the difference was not statistically significant (p = 0.196). Recurrence was uncommon in both groups (3.7% vs. 0%), and exploratory multivariable analysis revealed that CBM588 use was not independently associated with clinical cure. These findings do not establish a definitive benefit of adjunctive CBM588 therapy and should be interpreted cautiously as hypothesis-generating given the limited sample size and non-significant result.

BioTech doi: 10.3390/biotech15020043

Authors: Solomon Habtemariam

Highlighting its pivotal role in modern pharmacology, the gut microbiome is emerging as a key determinant of drug efficacy, toxicity, and bioavailability. This review proposes the Gut Microbiome Dependency Continuum, a four-layer framework describing progressively deeper levels of microbiome involvement in drug discovery and therapeutic function. The first layer, intact functional microbiome-dependent therapeutics and includes interventions such as faecal microbiota transplantation and defined microbial consortia. The second layer, microbiome-modulated approved drugs include widely used therapeutics whose pharmacokinetics or pharmacodynamics are strongly influenced by microbial metabolism. Examples include metformin, irinotecan, levodopa, and digoxin, where gut microbial interactions influence efficacy, toxicity, and inter-individual variability in treatment outcomes. The third layer, microbiota-transformable natural products, encompasses dietary and plant-derived compounds such as polyphenols, ginsenosides, alkaloids, fibres, isoflavones, lignans, and glucosinolates. Their biological activity depends on microbial biotransformation into bioactive metabolites. The fourth layer, engineered microbiome therapeutics, includes synthetic biology approaches such as programmable microbial systems, engineered probiotics, CRISPR-based microbiome editing, and microbiome-responsive drug delivery systems. It also includes synthetic microbial consortia, enabling targeted sensing, therapeutic delivery, and ecological reprogramming of gut microbial communities. Altogether, these layers define a continuum in which the gut microbiome evolves from a passive modulator to an essential metabolic organ and ultimately a programmable therapeutic platform. The article provides an integrated framework for microbiome-informed drug discovery. It also supports the development of precision, ecology-aware, and engineered microbial therapeutics.

BioTech doi: 10.3390/biotech15020042

Authors: Rita Domingues José C. M. Pires

Understanding biological communities is essential for elucidating ecosystem structure and function. Metabarcoding based on ribosomal RNA (rRNA) genes, particularly 16S and 23S, is widely used to characterise bacterial and microalgal communities. However, analysing high-throughput sequencing data generated by platforms such as the Illumina MiSeq remains challenging due to fragmented bioinformatics tools, complex parameterisation, and limited accessibility for non-specialist users. In this study, a comprehensive and user-friendly bioinformatics pipeline is proposed for the analysis of 16S and 23S paired-end metabarcoding data. The workflow integrates all critical processing steps, including read merging, primer and adapter trimming, quality filtering, dereplication, chimaera removal, and clustering into Operational Taxonomic Units (OTUs). Taxonomic assignment is performed using curated reference databases, namely EZBioCloud for bacterial communities and µgreen for microalgae. The pipeline was developed in Python 3.11 and incorporates validated tools such as VSEARCH and Cutadapt, ensuring robustness and computational efficiency. Additionally, modules for alpha and beta diversity analysis are included to support comprehensive ecological interpretation. The main novelty of this work lies in providing a unified, GUI-based framework that enables the standardised processing of dual-marker (16S/23S) metabarcoding data within a single environment. In its current implementation, SOMBA supports the analysis of each marker through separate but harmonised workflows, ensuring consistency in parameterisation, processing steps, and output structure. This approach provides an accessible and standardised solution that bridges the gap between raw sequencing data and reliable biological insights, supporting applications in environmental microbiology and biotechnology.

BioTech doi: 10.3390/biotech15020041

Authors: David Kwame Amenorfenyo Wenquan Zheng Zhe Cao Junhao Huang Zitong Deng Jiacheng Ruan Feng Li Hua Xiao

This study assessed the effects of different intensities of broad-spectrum white LED light (PAR range: 415–748 nm) on growth, fucoxanthin accumulation, and fatty acid composition of Isochrysis galbana. This study classified light intensity into three categories based on the white LED light source: high (HL, 150 μmol·m−2·s−1), medium (ML, 80 μmol·m−2·s−1), and low (LL, 30 μmol·m−2·s−1). The results showed that biomass concentration was optimized under high light intensity (HL, 150 μmol·m−2·s−1), whereas low light (LL, 30 μmol·m−2·s−1) yielded the highest fucoxanthin concentration (71.15 mg/L on day 12) and the only positive volumetric fucoxanthin productivity (3.14 mg/L/d) among the three treatments tested. The results further showed that low light (LL, 30 μmol·m−2·s−1) produced maximum cell density (10.08 × 106 cells/mL) and the most polyunsaturated fatty acids (PUFAs), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which constituted 1.93% and 22.47% of total fatty acids, respectively. This study demonstrates that low-intensity (LL, 30 μmol·m−2·s−1) white LED light supports the maximum co-production of valuable metabolites in I. galbana, establishing a scientific basis for scaling up I. galbana cultivation for nutraceutical and aquafeed applications.

BioTech doi: 10.3390/biotech15020040

Authors: Emmeran Bieringer Lisa Pütthoff Arne Zimmermann Ekaterina Burkhanova David Mijačević Armin Ehrenreich Wolfgang Liebl Dirk Weuster-Botz

Mucic acid (MA) is used as a chelating agent or as a building block for bio-based polymers. MA can be produced by regioselective oxidation of D-galacturonic acid (GA). Gluconobacter oxydans is known for the partial oxidation of various substrates via membrane-bound dehydrogenases. As the wild-type strain shows only low oxidation activity towards GA, the engineered multideletion strain G. oxydans BP9.1 pta-mGDH, overexpressing a membrane-bound glucose dehydrogenase from Pseudomonas taetrolens, was used in buffered whole-cell batch biotransformations with GA as the sole substrate. Initial cell-specific MA formation rates elevated with rising educt concentrations up to 63 g L−1. At pH 4, full GA conversion was only achieved with an initial GA concentration of 10 g L−1. Complete conversion of 94 g L−1 of GA was achieved at pH 5 with 3.4 g L−1 of G. oxydans BP9.1 pta-mGDH within 48 h, resulting in >100 g L−1 of MA, corresponding to a yield of >99% (mol/mol). Isolation of MA (purity > 90%) was achieved after cell separation, followed by cooling crystallization and drying, with a yield of 94%. Complete, full-yield GA conversion using non-growing cells of engineered G. oxydans in simple phosphate buffer yielded high product concentrations and enabled simple, high-yield product isolation, thus resulting in cost-effective and sustainable bioproduction of MA.

BioTech doi: 10.3390/biotech15020039

Authors: Tamiris C. Sardinha Philipe P. L. Pereira Tamires A. R. Gomes Bruna de A. C. F. Mendes Stefani M. Ferreira Leonila E. R. Raspantini Cristina de O. Massoco André T. Gotardo

Repeated blood collection in long-term studies in rodents is challenging and may compromise animal welfare. Rabbits represent a robust alternative model for longitudinal studies due to their larger blood volume and ease of repeated sampling. However, standardized assays to assess rabbit immune function remain scarce. This study presents standardized and optimized flow cytometry–based protocols for evaluating oxidative burst, phagocytosis, and lymphocyte proliferation in rabbits. Oxidative burst and phagocytic activity were analyzed in heparinized whole blood using DCFH and fluorescently labeled Staphylococcus aureus. Lymphocyte proliferation was assessed in CFSE-labeled PBMC stimulated with ConA. Flow cytometric analysis enabled simultaneous quantification of reactive oxygen species (ROS) generation, phagocytic uptake, and CFSE dilution modeling. Following stimulation with S. aureus, rabbit heterophils exhibited ROS production in a median of 90.4% (IQR: 84.8–92.9%) of gated cells, with a median phagocytic uptake of 23.6% (IQR: 13.4–27.2%). PMA-stimulated cells showed near-complete oxidative burst (median 99.1%, IQR: 98.7–99.5%), confirming their functional similarity to mammalian neutrophils. Lymphocytes exhibited measurable proliferative responses to ConA, validating PBMC-based assays for adaptive immune assessment. These standardized methods offer a framework for investigating innate and adaptive immune functions in rabbits, contributing to immunotoxicological and safety evaluation studies.

BioTech doi: 10.3390/biotech15020038

Authors: Mona Pißarreck Kristina Katsoutas Jörn Stitz

Objectives: Membrane-enveloped virus-like particles (VLPs) constitute a versatile vaccine platform allowing for the display of heterologous viral surface antigens. The density of displayed antigens is paramount for the efficient elicitation of a strong cellular and humoral immune response. SARS-CoV-2 spike protein variants with engineered cytoplasmic tails (CTs) were generated to enhance decoration efficiency on the surface of VLPs formed by the HIV core protein Gag. These HIV (SARS-CoV-2) chimeric particles serve as a vaccine component prototype. Methods: Spike variants were first analyzed for cellular and surface expression as well as incorporation into extracellular vesicles (EVs) and VLPs using flow cytometric analysis and Western blot analysis. Receptor binding, fusogenicity, i.e., mediating the fusion of spike-positive with receptor-containing membranes, and the proteins’ potential to mediate lentiviral vector gene transduction into susceptible target cells was examined by employing syncytia-formation assays and vector titration experiments. The display of a neutralization-sensitive epitope was examined utilizing immuno-precipitation using a neutralizing antibody. Results: All four variants were shown to be cell-surface expressed, to recruit the cognate receptor, to mediate membrane fusion and cell entry of lentiviral pseudotype vector particles and to decorate VLPs and EVs. However, the spike variant encompassing a truncated CT derived from the gibbon ape leukemia virus (GaLV) transmembrane (TM) envelope protein was most efficiently incorporated into HIV Gag-formed VLPs. All variants exposed a neutralization-sensitive epitope in the receptor binding domain. Conclusions: Engineering of the CTs of viral surface antigens can enhance VLP decoration, while required functionality of the ecto-domain such as receptor recognition, fusogenicity and neutralization-sensitive epitope presentation are not abrogated. This indicates the preservation of the structural integrity of the antigen required to elicit a neutralizing humoral immunity upon vaccination. The identified truncated CT of GaLV TM may be of utility to improve the incorporation of other viral surface antigens into a variety of membrane-enveloped VLPs derived from a range of different parental viruses.

BioTech doi: 10.3390/biotech15020037

Authors: Rania H. H. Ahmed Mahmoud E. Soliman Sherif F. Hammad Hesham S. M. Soliman Ahmed Abdel-Mawgood

Curcumin can behave potently as an anti-inflammatory agent, but unfortunately, it suffers from a detrimental aqueous solubility and a limited bioavailability, which hinders its usage in clinical applications. Formulating a polymeric nanosized biocompatible system can increase the delivered curcumin fraction intra-articularly for inflammatory diseases. Poly(glyceryl adipate) nanoparticles were synthesized using a polycondensation process yielding six different polymers, which was followed by a preparation of different curcumin-loaded formulations using the nanoprecipitation method. The selected formulation exhibited a particle size of 115.8 ± 10.9 nm, a mean PDI of 0.191 ± 0.038, a zeta potential of −22.9 ± 1.5 mV, and an EE of about 95%. This formulation was subjected to physicochemical characterization, including TEM, FTIR, DSC, and an in vitro release study. The stability was assessed in the presence of sodium sulfate at different concentrations, and the long-term stability was assessed through three months of storage. The cytocompatibility was evaluated by an MTT assay on normal and cancerous cell lines, revealing no significant cytotoxicity, and RT-qPCR was done in order to point out the anti-inflammation effect of the produced nanoparticles. Finally, a molecular docking study was held against Bruton’s tyrosine kinase. The formulation’s findings demonstrate the potential of nanoparticles, providing a basis for further in vivo studies targeting arthritis therapy.

BioTech doi: 10.3390/biotech15020036

Authors: Inês S. Almeida Eva M. Salgado António M. A. Ferreira José C. M. Pires

This study evaluates the oscillatory frequency and amplitude in an oscillatory flow reactor with smooth periodic constrictions (OFR-SPC) for the cultivation and harvesting of Chlorella vulgaris fed with an air stream with 5% (v/v) CO2. Their effect on biomass productivity, CO2 capture, nutrient removal, and sedimentation kinetics was assessed. Cultures were tested at frequencies of 0.5–2.5 Hz and amplitudes of 6–18 mm. At 2.5 Hz|6 mm, the system achieved the maximum biomass concentration (592 mgDW L−1), productivity (5.36 mgDW L−1 h−1), and CO2 fixation (8.34 mg L−1 h−1) as well as complete nitrogen removal and near-complete phosphorus removal (100% and 91%, respectively). Complete sedimentation occurred at 0.5 Hz|6 mm, with kinetics described by the Gompertz model (k = 4.60 h−1), confirming the feasibility of low-cost biomass recovery. Additionally, zeta potential positively influenced sedimentation but negatively affected productivity. Statistical analyses confirmed oscillation frequency and amplitude as key factors, establishing the OFR-SPC as a promising technology for microalgae-based efficient CO2 capture, nutrient removal, and low-cost biomass harvesting.

BioTech doi: 10.3390/biotech15020035

Authors: Ahmed Kanfoud Pascal Gerkens Marie Bastin Laurent Rondia Florian Ceulemans Karim Donnay Bertrand Debuisseret Thomas Cornet Gael de Lannoy Thibault Helleputte

Spectral monitoring combined with chemometrics models resulting from machine learning approaches allows cell culture to be monitored almost in real time. This process analytical technology offers to drastically reduce the amount of hands-on time and laboratory testing needed to monitor this crucial biomanufacturing step. In this article, we propose a method to anticipate future spectra. The method is based on extrapolation of the spectra in a reduced-dimensionality space, followed by retroprojection in the original space. Passed to regular chemometrics models already fitted, these anticipated spectra enable predictive cell culture monitoring up to several dozen hours with satisfactory quality. This anticipation paves the way for course-correction and enhanced operations such as reduced need for night shifts.

BioTech doi: 10.3390/biotech15020034

Authors: Hannah Morris Zoe Coombes Zeinab El Dor Valerie J. Rodrigues Alla Silkina Pietro Marchese Mary Murphy Jessica M. M. Adams Frank Barry Claudio Fuentes-Grünewald Walid Rachidi Deyarina Gonzalez

Marine macroalgae, microalgae, and associated microorganisms are increasingly recognised as valuable sources of bioactive compounds with applications across biotechnology and health. The environmental and ecological conditions they inhabit shape their metabolite diversity, leading to the production of high-value compounds such as sulphated polysaccharides, lipids, pigments, phenolics, and peptides. These compounds exhibit conserved biological activities that underpin potent antioxidant, anti-inflammatory, cytotoxic, and pro-regenerative effects with strong potential for translation. Although external factors drive rich metabolite diversity, continual variation can also lead to translational constraints including heavy-metal accumulation, inconsistency in extract composition, and regulatory complexity. This review examines the environmental drivers of metabolite diversity and the functional potential of bioactives derived from marine algae. We focus on their translational application within four areas of growing interest: nutraceuticals, cosmetics, regenerative medicine, and oncology, where emerging evidence suggests their promise as next-generation bioactive ingredients and therapeutic leads. In addition, insights from Irish and Welsh Small and Medium Enterprises (SMEs) are collated to identify key bottlenecks in commercialisation and the requirements for effective marine biodiscovery pipelines. We consider the importance of controlled cultivation, standardised analytics, preclinical testing platforms, and collaborative innovation ecosystems and highlight the need for coordinated scientific, technical, and regulatory advances to unlock the full translational potential of marine-derived compounds.

BioTech doi: 10.3390/biotech15020033

Authors: Marcos Pérez-Losada Manuel Aira Jorge Domínguez

Sewage sludge management poses major environmental challenges due to increasing production and concerns about contaminants and microbial risks. Vermicomposting offers a sustainable biological treatment, yet the extent to which different earthworm species shape microbial outcomes remains poorly understood. Here, we examined how gut transit by three epigeic (Eisenia andrei, E. fetida, and Dendrobaena hortensis) and two anecic (Lumbricus friendi and L. terrestris) earthworm species alters bacterial and fungal communities in fresh sewage sludge. Using 16S rRNA and ITS amplicon sequencing combined with multivariate, differential-abundance, and functional prediction analyses, we compared sludge and earthworm cast bacteriomes and mycobiomes. Earthworm gut transit caused pronounced species-specific restructuring of bacterial and fungal community composition, diversity, and functional profiles, with clear separation between sludge and cast communities. Functional analyses indicated coordinated shifts in bacterial metabolic potential and fungal trophic modes consistent with enhanced biosynthetic and decomposer functions. Pathogen profiles were reshaped in a host-dependent manner, with low overall abundances and selective changes rather than uniform suppression. These findings demonstrate that vermicomposting outcomes depend strongly on earthworm species and microbial kingdom, highlighting the importance of earthworm lifestyle diversity when evaluating the ecological safety and agronomic potential of sludge-derived amendments.

BioTech doi: 10.3390/biotech15020032

Authors: Marisol Cruz Requena Miguel A. Medina-Morales Paola Cano Reséndez Leonardo Sepúlveda Torre Thelma K. Morales Martínez Karina Reyes Acosta Catalina Hernández Torres Miriam Desiree Dávila Medina

Post-harvest diseases caused by phytopathogenic microorganisms generate significant economic losses, particularly in tomato crops affected by Alternaria solani. This study evaluated the effectiveness of cold plasma combined with a bioextract of Bacillus vallismortis as a biological strategy to extend tomato shelf life. In vitro antagonism assays were performed by confronting B. vallismortis against A. solani. Additionally, shelf-life tests were conducted on tomatoes treated with Bacillus cells and Bacillus cell-free bioextract (BCFB), followed by inoculation with A. solani spores, assessing incidence, severity, weight loss, and microbiological parameters over time. Subsequently, tomatoes were treated with cold plasma in combination with BCFB and reevaluated. Results showed significant antagonistic activity, with B. vallismortis and BCFB inhibiting A. solani by 75% and 50%, respectively. In untreated tomatoes, BCFB reduced disease incidence to 66.66% and severity to scale 2, compared to 100% incidence and scale 5 severity in controls. The combined treatment with cold plasma and BCFB showed the highest effectiveness, completely inhibiting A. solani (0% incidence, scale 0 severity), with tomatoes remaining healthy after 25 days. These findings demonstrate that cold plasma combined with B. vallismortis represents an effective and sustainable alternative for controlling post-harvest phytopathogens and extending tomato shelf life.

BioTech doi: 10.3390/biotech15020031

Authors: Odilon Souza Leite-Barbosa Debora Cristina da Silva Santos Cláudia Carnaval de Oliveira Pinto Fernanda Cristina Fernandes Braga Marcia Gomes de Oliveira Marcelo Ferreira Leão de Oliveira Valdir Florêncio da Veiga-Junior

Background: The transition from fossil-derived polymer additives to renewable alternatives is essential to mitigate environmental persistence and ensure chemical safety within the plastics industry. This review provides a comprehensive overview of recent developments in bio-based functional additives and their integration into circular economy frameworks. Methods: Following PRISMA guidelines, a systematic literature search was conducted using the Scopus database for studies published between 2023 and 2026. Search terms targeted bio-based plasticizers, flame retardants, antioxidants, and compatibilizers. Studies were screened against predefined inclusion criteria, specifically focusing on experimental validation in polymer matrices, while data mining was employed to map emerging research fronts. Results: From an initial 996 records, 54 studies were selected after removing duplicates and ineligible articles. The findings highlight a paradigm shift from passive physical fillers toward active, multifunctional macromolecular agents. Recent literature demonstrates that targeted molecular interventions, such as phosphorylated lignin and biomimetic structures, can resolve trade-offs between ductility and thermal stability at low loadings (<5 wt%). Synthesis routes, performance outcomes, and end-of-life trajectories for each additive class are summarized. Conclusions: Bio-based additives have evolved from simple substitutes into strategic tools for the molecular programming of sustainable polymers. Although challenges regarding scalability and high-temperature processing persist, their integration into circular economy strategies establishes a clear roadmap for next-generation bioplastics.

BioTech doi: 10.3390/biotech15020030

Authors: Lelde Grantina-Ievina Nils Rostoks

Genetically modified (GM) plants have been commercially grown for 30 years, and their acceptance depends on a thorough risk assessment. Environmental Risk Assessment (ERA) evaluates potential impacts of releasing GM plants into the environment, whether through cultivation or import for food, feed, and processing. A key component is assessing potential gene flow to crop wild relatives or non-GM crops. For gene flow to significantly affect the environment, transferred genes must provide a selective advantage. Since most GM plants are engineered for herbicide tolerance, insect resistance, or stacked traits, evaluating such advantages is relatively straightforward. New genomic techniques (NGTs) can generate plants with a wider range of traits, including tolerance to biotic and abiotic stress. Although still considered GM in the EU, their genomic changes can complicate detection, identification, and ERA, especially when such traits may offer advantages under stress conditions. This scoping review focuses on gene flow in two crops: oilseed rape (canola) (Brassica napus L.) and potato (Solanum tuberosum L.). In canola, transgene movement can increase weediness, fitness, herbicide resistance, or genetic diversity in feral or related populations. Gene flow in potato is less studied, with concerns centered on contamination risks in the Andean diversity center. Limited data exist for NGT plants, though many are expected to resemble conventionally bred varieties, suggesting comparable environmental impacts.

BioTech doi: 10.3390/biotech15020029

Authors: Levi Isai Solano-González Raúl Mendoza-Báez Ricardo Agustín-Serrano José Isrrael Rodríguez-Mora Marco A. Morales

In this work, the electronic, thermochemical, and vibrational characterization of the drug delivery system formed by clusters of selenium (Se6 allotrope) and 5-fluorouracil (5-FU) are studied, based on density functional theory (DFT) calculations. Computational calculations were performed using the B3LYP functional and the 6-31G(d,p) base set, considering an aqueous medium through the CPCM solvation model. We propose evaluating two different interaction modes based on experimental observations: Se–H(N) (through the amino groups of 5-FU) and Se–O(C) (through the carbonyl oxygen of 5-FU). All complexes proved to be energetically stable, exhibiting chemisorption as their adsorption process. Analysis of adsorption energy and thermodynamic parameters indicates that both interaction pathways are equally viable, which agrees with previous experimental findings. The theoretical FT-IR spectra of these complexes also coincide with the experimental results. Furthermore, global molecular descriptors show that the stability of the selenium carrier is not affected by post-functionalization, which is desirable for more controlled drug delivery systems.

BioTech doi: 10.3390/biotech15020028

Authors: Jimmy Carter Osei Mei-Han Chen Tim A. D. Smith

Radiotherapy (RT) is one of the main treatments for breast cancer, but response varies between patients. Tumour hypoxia and intrinsic radiosensitivity are major determinants of response to RT. Using TCGA-BRCA, a 563-gene hypoxia meta-signature was built by combining curated hypoxia gene sets from MSigDB with published hypoxia metagenes (Buffa, Winter, Elvidge, Fardin, and related sets). After Cox screening and penalised regression, a simple three-gene hypoxia score (CP, GPC3, STC1) was derived. In parallel, based on DSB-repair factors highlighted by Mladenov et al. as key regulators of intrinsic radiosensitivity, a four-gene radiosensitivity (RS) signature (ATR, RPA2, BLM, MRE11A) was trained using only RT-treated patients. In TCGA, both signatures were prognostic and showed significant interaction with RT status in Cox models. The hypoxia score was strongly associated with worse outcomes in RT-untreated patients, but this effect was much weaker in RT-treated patients (Hypoxia × RT HR = 0.009, p = 0.044). The RS score showed a similarly strong interaction with RT (RS × RT HR = 0.011, p = 0.003). When we combined both signatures into one interaction model, it gave the best performance (C-index = 0.785), and both interaction terms stayed independently significant. The hypoxia score was then validated externally in METABRIC (N = 1979; 1143 events), where it remained associated with overall survival, although more weakly than in TCGA (HR = 1.34, 95% CI: 1.10–1.63; p = 0.0042). Overall, these results suggest that hypoxia and DSB-repair capacity capture two complementary sides of radiosensitivity and RT-modified survival patterns, and they support further prospective testing and validation in independent datasets with strong RT annotation.

BioTech doi: 10.3390/biotech15020027

Authors: Sadith Mosquera Jason A. Rosenzweig

Lubricating oil (LO) is manufactured in various formulations for different applications. The inappropriate disposal of petroleum hydrocarbons can increase soil contamination, promoting deleterious environmental and human health impacts. More specifically, following prolonged exposure, LO contaminants are known to have carcinogenic and neurotoxic effects in humans. Bioremediation provides an effective and attractive strategy to expedite the clean-up processes of LO contaminants. We isolated and identified environmentally adapted strains of Pseudomonas aeruginosa, Pseudomonas putida, and Proteus vulgaris from Houston watershed bayou soils. Interestingly, all three exhibited increased resistance, vis-a-vis surrogate strains, to various antibiotic challenges (of chloramphenicol, tetracycline, kanamycin, penicillin, streptomycin, etc.) and increased biofilm formation ranging from 1.6 to 6.7-fold. In fact, all three environmental strains were significantly better at producing enhanced biofilm formation in the presence of spent LO rather than clean LO as well as outproducing biofilm made by the surrogate strains. Finally, the environmental isolates P. aeruginosa, P. putida, and P. vulgaris demonstrated an enhanced ability to sequester clean (2-, 2.5- and 1.14-fold) and spent (1.4-, 1.5, and 1.2-fold) LO when compared to their commercially acquired surrogate reference strains. Our three environmentally isolated organisms from Houston watershed soils appeared to be environmentally adapted to tolerate LO exposures. In the presence of LOs, all three environmentally isolated strains exhibited enhanced growth, enhanced biofilm production, and improved bioaccumulation of LOs relative to commercial reference strains. Taken together, environmentally adapted organisms can promote the bioremediation of contaminants threatening our environment and, potentially, human health.

BioTech doi: 10.3390/biotech15020026

Authors: Selena del Rocío Martínez-Betancourt Jorge Cadena-Iñiguez Laura Araceli López-Martínez Janet María León Morales Ramón Marcos Soto-Hernández Gerardo Loera-Alvarado Víctor Manuel Ruiz-Vera Concepción López-Padilla

Yucca decipiens is a native species from arid and semi-arid regions with emerging nutritional and biotechnological potential. This study evaluated its proximate composition, elemental profile determined by inductively coupled plasma mass spectrometry (ICP-MS), and growth performance under nursery conditions. Proximate analysis revealed a high dietary fiber content in leaves (58.93%) and higher carbohydrate levels in stems (28.83%). Free amino acid content was significantly higher in stems (2.75 g histidine equivalents kg−1) than in leaves (1.76 g kg−1). Multi-elemental profiling (63 elements) showed organ-specific accumulation patterns, with essential macro- and micronutrients predominantly concentrated in leaves, including potassium (28,334 ppm) and calcium (15,345 ppm), while iron was the most abundant trace element in stems (1253 ppm). Principal component analysis (PCA) revealed clear organ-specific mineral partitioning between leaves and stems, indicating differentiated physiological roles and potential selective biomass utilization. Growth assessment conducted over a two-year period demonstrated steady biomass accumulation and good adaptive performance under nursery conditions. Overall, the results highlight the emerging nutritional and agroindustrial relevance of Yucca decipiens for applications in semi-arid environments.

BioTech doi: 10.3390/biotech15010025

Authors: Anna Vesnina Violeta Le Svetlana Ivanova Anna Frolova Irina Milentyeva Victor Atuchin Alexander Prosekov

In this work, Filipendula ulmaria (L.) Maxim, a perennial herbaceous plant from the Rosaceae family, was considered a novel source of obtaining rutin for pharmaceutical purposes. Rutin was extracted from the plant parts collected in the flowering summer period and dried at 40 ± 3 °C. The process was carried out using the ethanol extraction and fractionation of extracted compounds, and it yields the 95 wt% purity crystalline product. The phase composition of the extracted rutin was verified by the XRD analysis and NMR measurements. It was found that 2.85% of rutin could be extracted from Filipendula ulmaria, which is 1.2 times higher than the results of similar studies. The biological activity of the isolated rutin was tested on rats. It was established in vivo that the extracted rutin normalizes blood glucose levels (glucose and glycosylated hemoglobin), insulin resistance (HOMA-IR index) and reduces the severity of dystrophic changes in the liver caused by high-fat and high-carbohydrate diets. The introduction of rutin corrects lipid profile indicators (triglycerides, cholesterol, cholesterol fractions in lipoproteins and atherogenic indices), cytolysis indicators of hepatocytes, and liver steatosis (ALT, AST/ALT, triglycerides). Thus, the novel source of rutin opens the possibility for a wide use of this flavonoid in the food technology and pharmaceutical industry.

BioTech doi: 10.3390/biotech15010024

Authors: Samuel Nicacio Winston Umakanth Balasundaram Aboli Bhingarkar Daniel Cho Rashmi Ghayal Anup Datta Subhash V. Kapre

Different methods are used today for polysaccharide quantitation, including HPLC and various colorimetric assays. Among these, the anthrone-sulfuric acid assay (anthrone assay) is popular when the sample matrix is suitable, such as in purified polysaccharides and monovalent bulk conjugate components of glycoconjugate vaccines. While relatively safe, quick, and affordable, the anthrone assay requires significant operator time to complete and is not suited to high-throughput processing. Furthermore, the anthrone-sulfuric acid reagent presents a unique challenge to automation efforts due to its corrosive properties. Reported here is an automated anthrone assay via a liquid handling system (LHS). Twenty-three serotypes of pneumococcal (PNU) polysaccharide were quantified with the traditional anthrone assay and subsequently analyzed using the anthrone LHS method. The anthrone LHS method was evaluated for accuracy compared to the manual method and later validated according to ICH Q2 (R2) guidelines. To our knowledge, this is the first fully unattended and corrosion-mitigated anthrone assay validated under ICH Q2 (R2), capable of overnight batch operation. The developed assay can quantify polysaccharides with an accuracy of 81–115%, is precise to a coefficient of variation of <7.0%, and is linear between 30 and 650 µg/mL range (R2 ≥ 0.993). The assay can process eight samples per hour, can be utilized in overnight operation, and completes all pipetting, incubation, and data export steps automatically.

BioTech doi: 10.3390/biotech15010023

Authors: Shih-Yen Lo Chee-Hing Yang Yu-Ru Chan Yi-Tzu Chao Meng-Jiun Lai Hui-Chun Li

Transient DNA transfection is routinely used to study gene function and elucidate the regulation of biological pathways, and it is also widely applied in biotechnology for large-scale recombinant protein production. The results of recent studies involving mammalian cells have highlighted that competition for cellular resources during gene expression can bias data interpretation, directly affecting co-transfection experiments. In this study, our results showed that co-transfected plasmids markedly enhance transient—but not stable—expression of various reporter genes across different cell types. The enhancement of transient reporter gene expression by additional plasmid DNA occurs when these DNAs are co-delivered simultaneously and is unlikely to be mediated by cytokine induction. Furthermore, co-transfected plasmids were shown to upregulate transcription, but not translation, of the reporter gene during transient expression. Thus, the observed enhancement may result from competition between co-transfected plasmids and reporter constructs for cellular proteins that interact with transfected DNA, such as histones. Indeed, Pracinostat (SB939), an inhibitor of histone deacetylase, was able to enhance the transient expression of the reporter gene dose-dependently. Overall, this study provides insights that may facilitate improved transient expression of recombinant genes in biotechnological applications.

BioTech doi: 10.3390/biotech15010022

Authors: Constantinos H. Papadopoulos Dimitris Karelas Christina Floropoulou Konstantina Tzavida Dimitrios Oikonomidis Athanasios Tasoulis Evangelos Tatsis Ioannis Kouloulias Nikolaos P. E. Kadoglou

The rapid expansion of digital technologies and artificial intelligence (AI) has profoundly transformed cardiovascular imaging, enabling more precise, efficient, and reproducible assessment of cardiac structure and function. This narrative review summarizes recent advances in AI-driven methods across echocardiography, cardiac computed tomography, cardiac magnetic resonance, and nuclear imaging, with emphasis on image acquisition, automated quantification, and diagnostic and prognostic interpretation. We reviewed contemporary literature describing machine-learning and deep-learning applications for image reconstruction, segmentation, radiomics, and multimodal data integration. Current evidence demonstrates that AI improves image quality, reduces acquisition and analysis time, and enables automated, highly reproducible measurements of chamber volumes, function, tissue characterization, coronary anatomy, and myocardial perfusion, while facilitating advanced pattern recognition for differential diagnosis and risk stratification. Furthermore, digital platforms support remote acquisition, tele-echocardiography, and AI-assisted training of non-expert operators. Despite these advances, challenges remain regarding external validation, generalizability across vendors and populations, explainability, data governance, and regulatory compliance. In conclusion, AI and digital technologies are reshaping cardiovascular imaging by enhancing accuracy, efficiency, and accessibility, but their safe and effective clinical integration requires robust multicenter validation, transparent reporting, and ethical-legal frameworks that ensure trust, equity, and accountability.

BioTech doi: 10.3390/biotech15010021

Authors: Ivan Guryanov Sirina Sabirova Svetlana Batasheva Svetlana Konnova Arthur Khannanov Marianna Kutyreva Ekaterina Naumenko

The therapeutic potential of prodigiosin as a hydrophobic anticancer agent can be enhanced by various approaches, one of which is the loading of PG into extracellular vesicles. Drug distribution and stability in aqueous media play a crucial role in targeting and accumulation, thereby enabling the attainment of therapeutically effective drug concentrations. Extracellular vesicles are nano-sized, cell-derived vesicles with a lipid bilayer membrane. Extracellular vesicles can be utilized as drug carriers for both water-soluble and non-water-soluble therapeutic agents. We hypothesized that microvesicles could effectively address the current challenges of prodigiosin delivery. Several different techniques have been developed for fabricating extracellular vesicles. These include microvesicles induction by cytochalasin B treatment as well as cell cultivation in serum depleted media. In our study, prodigiosin, like cytochalasin B, demonstrated efficacy in microvesicles formation based on protein quantification and Nanoparticle Tracking Analysis. In addition, Nanoparticle Tracking Analysis showed that vesicles from mesenchymal stem cells are more stable under ultrasound exposure. Microvesicles encapsulating prodigiosin, compared to unmodified naïve ones, demonstrated slightly increased zeta potentials and hydrodynamic diameters, which probably contributed to better stability. We demonstrated that ultrasonic treatment for the loading of prodigiosin does not significantly increase the proportion of prodigiosin-positive microvesicles in comparison with microvesicles induced with prodigiosin; moreover, this method cannot be considered as optimal due to its disadvantages, such as particle aggregation. Prodigiosin-induced and prodigiosin-loaded microvesicles from mesenchymal stem cells were significantly smaller and less polydisperse in size. Overall, prodigiosin encapsulated in extracellular vesicles might be more suitable for medical and clinical applications compared to pure forms of PG due to their cell membrane compatibility.

BioTech doi: 10.3390/biotech15010020

Authors: Shuaibo Shi Ting Xiong Dong Wang Lingling Wei Lin Li Zhixin Li Yanfen Lyu

The study of protein–protein interactions (PPIs) is of significant importance for elucidating biological processes, clarifying pathological mechanisms, and promoting drug development. In this study, we proposed a method to predict PPIs based on protein sequence and gene sequence information, combined with convolutional neural networks (CNNs). First, we extracted three types of features from protein sequence: global physicochemical properties features of the protein sequence, local same type of amino acid position variation features, and protein evolutionary conservation features; simultaneously, we extracted single nucleotide frequency and positional features, dinucleotide frequency features, and trinucleotide frequency features from the corresponding gene sequence. During the feature extraction process, we employed the amphiphilic pseudo amino acid composition (APAAC) method to extract the global hydrophobicity and hydrophilicity features of the protein sequence; we defined a new mathematical descriptor—θ interval deviation product factor—to extract protein evolutionary conservation features from Position Specific Scoring Matrix (PSSM); we also defined a mapping function to map all nucleotides in the gene sequence onto a unit circle, and then extracted nucleotide positional features from the mapped points. Second, based on extracted features, we constructed a 36 × 32 sample feature grayscale map to represent a protein pair sample. Finally, we developed a CNN model to predict PPIs. Our method achieved superior results on four species test sets: an accuracy of 99.28% on the Saccharomyces cerevisiae dataset, 98.15% on the Drosophila melanogaster dataset, 98.62% on the Homo sapiens dataset, and 96.84% on the Mus musculus dataset, outperforming existing computational methods. Furthermore, we extended the application of this method to the prediction of protein–protein interaction networks and non-interaction networks, and also achieved promising results.

BioTech doi: 10.3390/biotech15010019

Authors: Christiana Bitrus Ademola Hammed Tawakalt Ayodele Niloy Chandra Sarker

Background/Objectives: The effects of thermal drying on the viability of beneficial microorganisms immobilized in biochar, as well as on biochar nutrient retention, remain insufficiently understood. This study aimed to evaluate how drying temperature influences the survival of hyper-ammonia-producing bacteria (HAB) immobilized on pine wood biochar and to assess the impact of subsequent storage on bacterial recovery and nutrient stability. Methods: Biochar was inoculated with HAB and subjected to drying at temperatures ranging from 40 to 60 °C. Following drying, samples were characterized and stored for 30 days. Microbial revival was assessed through reculturing, while changes in surface functional groups were analyzed using FTIR spectroscopy. Nutrient retention, particularly nitrogen content, was also evaluated. Results: Higher drying temperatures resulted in reduced immediate microbial revival during reculturing. However, samples exhibiting limited immediate recovery demonstrated enhanced revival after the 30-day storage period. FTIR analysis revealed that drying temperature modified the availability of surface functional groups associated with microbial attachment and activity. Nutrient analysis indicated only minor reductions in nitrogen retention in biochar dried at temperatures above 55 °C. Conclusions: Drying temperature significantly affects both the short-term survival and post-storage recovery of beneficial microorganisms immobilized in biochar. While elevated temperatures may initially suppress microbial activity, recovery potential during storage remains substantial. Optimizing drying conditions is therefore essential to balance microbial viability with nutrient retention in biochar-based formulations.

BioTech doi: 10.3390/biotech15010018

Authors: Angelo Michilli Cristian Bassi Farzaneh Moshiri Bruno De Siena Rosaria Marinaro Elisa Callegari Massimo Negrini Silvia Sabbioni

Despite the advent of immune checkpoint inhibitor-based regimens, sorafenib remains an important therapeutic option for patients with advanced hepatocellular carcinoma (HCC) who are ineligible for immunotherapy. However, its clinical efficacy is limited by the emergence of drug resistance, whose underlying molecular mechanisms remain incompletely understood. To investigate these mechanisms, we established a murine model of acquired sorafenib resistance and performed comparative RNA sequencing of sorafenib-sensitive versus -resistant Hep55.1C hepatoma cells. Transcriptomic profiling revealed a distinct resistance-associated signature comprising 1264 significantly deregulated genes (adjusted p < 0.03, fold change > 3.0). Pathway analysis and Gene Set Enrichment Analyses (GSEA) indicated a coordinated downregulation of metabolic and intercellular signaling pathways, accompanied by marked upregulation of redox-regulatory, mitochondrial and cellular stress-response programs. Genes transcriptionally regulated by nuclear factor erythroid 2-related factor 2 (NRF2) including Gpx4, Txn1, Txnrd1, Hmox1, Fth1, Taldo1, Phgdh, and MafG, involved in antioxidant defense, ferroptosis suppression and metabolic rewiring, were all upregulated in resistant cells. Pharmacological inhibition of NRF2 activity using brusatol restored sensitivity to sorafenib, functionally implicating NRF2-dependent pathways in the maintenance of the resistant phenotype. These findings demonstrate that acquired sorafenib resistance in HCC is associated with a stable NRF2-driven transcriptional and metabolic reprogramming that enhances antioxidant capacity, suppresses ferroptosis and promotes tumor cell survival. Targeting NRF2-regulated redox metabolism may therefore represent a promising strategy to overcome therapeutic resistance in HCC.

BioTech doi: 10.3390/biotech15010017

Authors: Viola Papini Alessandra Benigno Domenico Rizzo Salvatore Moricca

Species of the genus Phytophthora are among the most detrimental plant pathogens globally, representing a significant threat to global agriculture, horticulture, and forestry. These zoosporic oomycetes have historically caused devastating outbreaks, including, just to mention a few, late blight of potato in Ireland; jarrah dieback of eucalyptus in Western Australia; ink disease of chestnut in Europe; sudden oak death and sudden larch death of coast live oak and tanoak in the Western US, and of Japanese larch in the UK. The environmental and ecological impacts of the diseases they cause result in significant economic costs that often have social repercussions. With the acceleration of globalization, enhancing the movement of plant material, in particular with the global live plant trade, the spread of Phytophthora to new, uncontaminated territories has intensified. Nurseries play a key role in the movement of these pathogens, the trade of contaminated stocks representing their major dissemination route. However valuable, conventional detection techniques, including baiting and direct isolation, are too slow and labour-intensive to meet current diagnostic requirements, particularly given the huge volumes of plants traded globally. This problem becomes even more acute when large volumes of potentially infectious plant material need to be processed in a short time frame, as it is often necessary to provide accurate and timely responses to interested parties. Early and precise detection is thus vital to avert outbreaks and mitigate long-term consequences. This review evaluates and contrasts the efficacy of novel detection methods against traditional approaches, emphasizing their significance in managing the escalating threat posed by Phytophthora spp. worldwide. Despite technological advances, critical challenges remain that limit the reliability and large-scale adoption of new diagnostic methods. Research still needs to bridge the gap between the laboratory and the field in terms of accuracy, sensitivity and diagnostic costs. Recent innovations focus on sensor technology and point-of-care (POC) devices for faster, more sensitive, and low-cost specific detection of Phytophthora spp. in plant matrices, water and soil. Enhancing diagnostic capabilities through these tools is crucial for protecting agricultural productivity, local economies, and natural ecosystems.

BioTech doi: 10.3390/biotech15010016

Authors: Ajay Thapa Shiyu Fu Joseph Sebastian Onita Basu Farah Hosseinian Utsav Sharma Dayanand Sharma Abid Hussain

Acidogenic fermentation is a promising biotechnology for converting organic wastes into carboxylic acid (CA), which has significant commercial value and diverse applications in the food, chemical, pharmaceutical, and cosmetic industries. However, major challenges such as limited substrate hydrolysis and lower CA production hinder further development of this biotechnology towards full-scale implementation. This review provides a comprehensive overview of the current status of acidogenic fermentation, focusing on substrate composition, inoculum, and reactor design, along with potential strategies to overcome reactor-specific limitations and enhance CA production. It was found that the substrate composition, particularly its carbohydrate, protein, and lipid contents, strongly influences both CA production and yield. Specifically, carbohydrate-rich substrates yield higher CA production compared to protein- and lipid-rich substrates. These substrates have been investigated in different reactor configurations for CA production. Among them, the leachate bed reactor and anaerobic membrane bioreactor have demonstrated superior performance, achieving higher CA production with acetic and butyric acids as the dominant CA composition. These reactors are generally operated using three types of inocula: aerobic and anaerobic inoculum, enriched inoculum, and rumen microorganisms. Interestingly, rumen microorganisms are effective in degrading complex substrates, whereas enriched inoculum accelerates hydrolysis and acidogenesis processes within a shorter fermentation time. The findings presented herein will provide valuable information for addressing the challenges associated with acidogenic fermentation and lay the foundation for future research aimed at upscaling this biotechnology to a commercial scale.

BioTech doi: 10.3390/biotech15010015

Authors: Haifan Li Lili Zhang Guodong Wang Tanjun Zhao

Carotenoid-based pigmentation is crucial for the ornamental and commercial value of the cherry shrimp (Neocaridina denticulata sinensis). While several genes are known to influence carotenoid metabolism, the genetic basis for specific color strains remains largely unexplored. Here, we functionally characterized NinaB-like, a homolog of a carotenoid oxygenase, in cherry shrimp pigmentation. We employed qPCR to gain gene expression profiles, utilized RNAi technology to analysize the relation between its expression level and carotenoid accumulation, and performed GT-seq to identify genotypes of different color strains. Significant differential expression of NinaB-like was observed not only across distinct color strains but also during embryonic development of cherry shrimp (p < 0.05), peaking at the red strain and post-larval stage of cherry shrimp. RNA interference-mediated knockdown of NinaB-like resulted in a marked increase in red pigment deposition at the metanauplius and pre-zoea stages, confirming its role as a negative regulator of carotenoid accumulation. Importantly, we identified two tightly linked, non-synonymous SNPs (927C > A and 935A > C) within the NinaB-like coding region that exhibited a strong association with body color. Our study provides the first functional evidence that NinaB-like is a negative regulator of carotenoid degradation and a major genetic determinant for body color in cherry shrimp, providing new insights for genetic breeding and biological research.

BioTech doi: 10.3390/biotech15010014

Authors: Shafqat Shabir Md. Shahadat Hossain Lucie Egly Gizem Yalkin Franco H. Falcone

Human embryonic kidney (HEK293) cells are a widespread choice for recombinant protein expression. To optimise yields, the hydrolysate Tryptone N1 (TN1) is commonly added post-transfection. TN1 is obtained by controlled enzymatic digestion of casein. As an animal by-product, TN1 faces stricter regulations during cross-country shipments than plant-based products. This raises the question of whether plant-derived peptides are a suitable alternative to TN1. Using polyethyleneimine (PEI) as a cationic polymer, we transfected HEK293-6E cells grown in suspension in serum-free medium and divided the transfectants into four groups (each in triplicate). Two plant-based hydrolysates each derived from pea and broad bean were compared with TN1 and a no-hydrolysate control group. We monitored the cultures for total cell numbers and viability at days 1, 4, and 5 post-transfection. Both plant-based hydrolysates and TN1 showed similar live cell percentages, in contrast to the no-hydrolysate control, which showed lower viability. Five days post-transfection, the expressed His-tagged protein, a tegumental antigen from the eukaryotic parasite Echinococcus granulosus, was retrieved from the serum-free culture supernatant, and the expressed recombinant protein was quantified. The linear ranges for the protein load on the stain-free blot and for the use of the fluorescent anti-His-Tag Alexa488 antibody were determined. Using these parameters, stain-free Western blotting and total protein normalization were performed. The plant-derived pea and broad bean hydrolysates reproducibly resulted in similar expression levels as animal-derived TN1; all three hydrolysates were better than no hydrolysate. We conclude that plant-derived hydrolysates are a suitable, more sustainable replacement for TN1.

BioTech doi: 10.3390/biotech15010013

Authors: Phuong Duy Nguyen Van Thi Pham Ha Thanh Nguyen Khoa Dang Dang Tu Tuan Tran Dai Lan Tran Thanh Duc Nguyen Thao Duc Le Xuan Hoi Pham Xuan Dang Tran Quyen Le Cao

This study investigates the functional role of OsHSBP1, a heat shock factor-binding protein, in regulating abiotic stress tolerance in rice, with the aim of enhancing climate resilience in the elite indica cultivar Bacthom 7 (BT7). Using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing, we generated transgene-free homozygous knockout lines targeting OsHSBP1 and evaluated their physiological, biochemical, and agronomic responses under heat stress. Mutant lines exhibited markedly improved tolerance to both stresses, with survival rates reaching 43–46% under heat stress, compared to near-zero in wildtype plants. Enhanced tolerance was associated with significantly increased catalase and peroxidase activities and reduced oxidative damage, including lower malondialdehyde content and decreased superoxide accumulation. Despite these stress-related advantages, the knockout lines showed minimal differences in key agronomic traits under normal growing conditions, with comparable plant height, tillering ability, grain yield, and amylose content relative to the wildtype. These results demonstrate that OsHSBP1 functions as a negative regulator of abiotic stress tolerance in rice, and its knockout enhances resilience without compromising yield potential. The study highlights OsHSBP1 as a promising target for precision breeding of climate-resilient rice cultivars.

BioTech doi: 10.3390/biotech15010012

Authors: Jonathan Puente-Rivera Stephanie I. Nuñez Olvera Ameyatzin Ereth Robles-Chávez Nayeli Goreti Nieto-Velázquez María Elizbeth Alvarez-Sánchez

Prostate cancer (PCa) is a leading cause of cancer-related mortality in men and is often characterized by aggressive growth and bone metastasis. Angiogenesis plays a central role in tumor progression and dissemination. This study aimed to explore the regulatory roles of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in angiogenesis and metastasis during PCa progression. Publicly available RNA-seq datasets were analyzed to identify differentially expressed miRNAs between metastatic (N1) and nonmetastatic (N0) PCa. Bioinformatic tools were used to reconstruct co-regulatory networks involving miRNAs, lncRNAs, and angiogenesis-related mRNAs. RT-qPCR was performed on serum-derived liquid biopsies from N0 and N1 patients and healthy controls to validate the key regulatory axes. Transcriptomic analysis revealed that miRNAs such as hsa-miR-183-5p and hsa-miR-216a-5p were upregulated in N1 PCa and associated with pro-angiogenic signaling, whereas hsa-miR-206 and hsa-miR-184, known for their anti-angiogenic functions, were downregulated. Network analysis identified the LINC00261–miR-206–HIF1A axis as the central regulatory module. RT-qPCR validation confirmed the significant downregulation of LINC00261 and miR-206, along with HIF1A overexpression in N1 samples compared to N0 and controls (p < 0.001), supporting in silico predictions. These findings highlight the role of ncRNA-mediated regulation of PCa angiogenesis and metastasis. The LINC00261–miR-206–HIF1A axis may serve as a promising noninvasive biomarker and potential therapeutic target. The integration of computational and experimental data provides a strong rationale for the further functional validation of advanced PCa.

BioTech doi: 10.3390/biotech15010011

Authors: Kenzhegul Bolatkhan Ardak B. Kakimova Bolatkhan K. Zayadan Akbota Kabayeva Sandugash K. Sandybayeva Aliyam A. Dauletova Tatsuya Tomo

The transition to low-carbon energy systems requires scalable and energy-efficient routes for producing liquid biofuels that are compatible with existing fuel infrastructures. This review focuses on bio-oil production from phototrophic microorganisms, highlighting their high biomass productivity, rapid growth, and inherent capacity for carbon dioxide fixation as key advantages over conventional biofuel feedstocks. Recent progress in thermochemical conversion technologies, particularly hydrothermal liquefaction (HTL) and fast pyrolysis, is critically assessed with respect to their suitability for wet and dry algal biomass, respectively. HTL enables direct processing of high-moisture biomass while avoiding energy-intensive drying, whereas fast pyrolysis offers high bio-oil yields from lipid-rich feedstocks. In parallel, catalytic upgrading strategies, including hydrodeoxygenation and related hydroprocessing routes, are discussed as essential steps for improving bio-oil stability, heating value, and fuel compatibility. Beyond conversion technologies, innovative biological and biotechnological strategies, such as strain optimization, stress induction, co-cultivation, and synthetic biology approaches, are examined for their role in tailoring biomass composition and enhancing bio-oil precursors. The integration of microalgal cultivation with wastewater utilization is briefly considered as a supporting strategy to reduce production costs and improve overall sustainability. Overall, this review emphasizes that the effective coupling of advanced thermochemical conversion with targeted biological optimization represents the most promising pathway for scalable bio-oil production from phototrophic microorganisms, positioning algal bio-oil as a viable contributor to future low-carbon energy systems.

BioTech doi: 10.3390/biotech15010010

Authors: Gregorio Peron

Oxidative processes influence several aspects of biology, from the subtle balance of redox signaling to the destructive cascade of oxidative damage associated with chronic disease and aging [...]

BioTech doi: 10.3390/biotech15010009

Authors: Elisa Callegari Angelo Michilli Farzaneh Moshiri Bruno De Siena Laura Gramantieri Massimo Negrini Silvia Sabbioni

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality. It usually arises in cirrhotic liver, where chronic inflammation and fibrosis create a tumor-permissive microenvironment. Dysregulation of microRNAs (miRNAs), particularly upregulation of the oncomiR miR-221 and loss of the tumor suppressor miR-199a-3p represent key drivers of liver carcinogenesis. The TG221 transgenic mouse, designed to overexpress miR-221 in hepatocytes, provides a relevant in vivo platform for mechanistic studies and for testing preventive and therapeutic approaches. The TG221 model recapitulates miR-221-driven tumorigenesis, including suppression of p27, p57 and Bmf. It is characterized by steatohepatitic injury and accelerated tumor formation after genotoxic challenge. In the cirrhotic CCl4-induced background, TG221 mice develop fibrosis and cirrhosis followed by dysplastic and malignant lesions, mirroring the natural history of human HCC. Metformin administered during early fibrosis prevented macroscopic tumor formation and suppressed PI3K/AKT/mTOR signaling. Anti-miR-221 and miR-199a-3p mimics reduced tumor burden, restored tumor-suppressive pathways and improved liver integrity, thus indicating feasible chemopreventive strategies. From a therapeutic point of view, miR-199a-3p replacement synergized with palbociclib and overcame sorafenib resistance. A miR-199a-3p-responsive oncolytic adenovirus achieved tumor-selective replication with minimal toxicity. This review highlights the importance of the TG221 transgenic mouse as a powerful model for studying miRNA-driven hepatocarcinogenesis and enables preclinical evaluation of RNA-based chemopreventive and therapeutic approaches. Metformin, miRNA inhibition, miRNA replacement and miRNA-guided viral therapies emerge as promising approaches for advancing precision prevention and treatment strategies in HCC.

BioTech doi: 10.3390/biotech15010008

Authors: Luis Jimenez

By analyzing Food and Drug Administration (FDA) enforcement reports from 2004 to 2025, we can determine the incidence of microbial contamination in non-sterile and sterile drugs in the United States of America and, at the same time, compare the trends and patterns over a period of 21 years to determine the distribution and frequency of microbial contaminants. The most common microorganisms detected from 2019 to 2025 were the mold Aspergillus penicilloides, with 17 citations for sterile products, followed by 16 citations for non-sterile products of Burkholderia cepacia complex (BCC) bacteria. Analysis from the last 21 years revealed the dominant microbial contaminants belong to the BCC, reaching a maximum level between 2012 and 2019. Some of the previous microbial contaminants, such as Salmonella and Clostridium, decline in the 2019–2025 period, with no notifications issued. S. aureus and Pseudomonas contamination persisted through the years but at very low levels. Gram-negative bacteria contaminated non-sterile drugs more frequently than Gram-positive. A worrisome trend continued with unacceptable levels of enforcement reports not providing any information on the identity of the microbial contaminant. New species of Bacillus and Acetobacter nitrogenifigens were responsible for a significant increase in non-sterile drug recalls. The main driver for sterile product recalls over a 21-year period is the lack of assurance of sterility (LAS) where major failures in process design, control, and operational execution were not conducive to the control of microbial proliferation and destruction. Enforcement data analysis identified the problematic trends and patterns regarding microbial contamination of drugs, providing important information to optimize process control and provide a framework for optimizing risk mitigation. Although the 21-year landscape demonstrated that some microbial contaminants have been successfully mitigated, others remain resilient. The emergence of new contaminants highlights the evolving nature of microbial risk. The consistent problem with LAS is not only a major regulatory violation but also a potential catalyst for the next major healthcare-associated outbreak.

BioTech doi: 10.3390/biotech15010007

Authors: Jing Sun Yingxue Xiao Lingling Xie Dan Qin Yue Zou Yingying Liu Yitong Zhai Minyi Zhang Tong Li Youjin Hao Bo Li

The tumor immune microenvironment (TIME) is closely involved in tumor initiation, malignant progression, immune escape, and response to immunotherapy. With the continued development of high-throughput sequencing technologies, transcriptomic approaches have become essential for examining the cellular and molecular features of the TIME. Bulk RNA sequencing offers tissue-level gene expression profiles and allows the estimation of immune cell composition through computational deconvolution. Single-cell RNA sequencing provides finer resolution, revealing cellular heterogeneity, lineage relationships, and functional states. Spatial transcriptomics (ST) retains the native anatomical context, making it possible to localize gene expression patterns and cell–cell interactions within intact tissues. These approaches, when considered together, have shifted TIME research from averaged measurements toward a more detailed and mechanistic understanding. This review summarizes the principles, applications and limitations of bulk, single-cell and spatial transcriptomic methods, highlighting emerging strategies for integrative analysis. Such multi-scale frameworks are increasingly important for studying immune dynamics and may contribute to the development of more precise biotechnological and immunotherapeutic strategies.

BioTech doi: 10.3390/biotech15010006

Authors: Ching Tse Kesen Ma

Iron-containing alcohol dehydrogenases (Fe-ADHs) from hyperthermophiles represent a distinct class of oxidoreductases characterized by exceptional thermostability, catalytic versatility, and unique metal-dependent properties. Despite considerable sequence diversity, Fe-ADHs share conserved motifs and a two-domain architecture essential for iron coordination and NAD(P)H cofactor binding. Physiologically, these enzymes are predicted to function primarily in aldehyde detoxification and redox homeostasis, with some also participating in fermentative alcohol production. Their remarkable stability and catalytic efficiency highlight their potential as robust biocatalysts for high-temperature industrial bioprocesses. This review presents a comprehensive comparative analysis of the biophysical, biochemical, and kinetic properties of Fe-ADHs, focusing on their thermostability, metal ion specificity, and catalytic mechanisms, as well as highlighting their potential for industrial biocatalytic applications.

BioTech doi: 10.3390/biotech15010005

Authors: Maryna Lutsenko Michela Ravelli Gregorio Peron

Medicinal honeys from Oceania have gained considerable attention due to their peculiar bioactive constituents and potential health applications. Apart from small molecules such as methylglyoxal and hydrogen peroxide, these honeys are rich in phenolic compounds, volatile terpenes, and other bioactive molecules, which collectively contribute to their antioxidant, antimicrobial, anti-inflammatory, and wound-healing properties. Recent studies have highlighted the distinctive composition of Oceania honeys such as Manuka (Leptospermum scoparium), Jarrah (Eucalyptus marginata), and Agastache (Agastache rugosa) from New Zealand and Australia, demonstrating variability in bioactivity depending on floral source, geographical origin, and processing methods. This review synthesizes the current knowledge on the chemical profiles of these honeys with a particular focus on bioactive compounds and distinctive markers, and evaluates their therapeutic potential. Emphasis is placed on the mechanisms underlying their bioactivities, as well as emerging clinical and preclinical evidence supporting their medicinal use. By consolidating recent findings, this work provides an updated perspective on the functional properties of Oceania honeys, underscoring their relevance as natural products with significant health-promoting potential.

BioTech doi: 10.3390/biotech15010004

Authors: Evgeniya Prazdnova Fadi Amirdzhanov Anuj Ranjan Radomir Skripnichenko

Bacillus-derived lipopeptides are known to possess diverse biological activities, including antimicrobial and anticancer properties, though the mechanisms of such effects at the molecular level remain incompletely understood. We investigated whether non-ribosomal peptide metabolites from Bacillus can directly interact with transmembrane receptors implicated in oxidative stress regulation and cancer progression (NOX4, EGFR, PDGFR, and OCTN2) using molecular docking and 200 ns molecular dynamics simulations of 11 lipopeptide metabolites. Molecular docking revealed several strong ligand–protein interactions, with plipastatin and fengycin emerging as lead compounds demonstrating the highest binding affinities to multiple receptors. For NOX4, iturin D showed the strongest docking score of −7.85 kcal/mol. Fengycin demonstrated a high docking score of −7.38 kcal/mol for PDGFR and −8.1 kcal/mol for EGFR. Plipastatin showed the strongest docking scores of −11.12 kcal/mol for EGFR and −8.7 kcal/mol for OCTN2. Molecular dynamics simulations confirmed complex stability for these lead compounds, with protein RMSD remaining stable at ~1.5 Å and ligand RMSD between 1.9 and 6 Å over 200 ns. Our findings suggest that plipastatin and fengycin may act as modulators of key receptors involved in oxidative stress and cancer-related signaling. However, those in silico predictions require experimental validation. This work provides the first computational evidence of potential lipopeptide–receptor interactions and establishes a foundation for future experimental investigation of probiotic-derived therapeutics.

BioTech doi: 10.3390/biotech15010003

Authors: Miho Takemura Takashi Maoka Norihiko Misawa

Torularhodin is the monocyclic C40 carotenoid with the β-ring and a terminal carboxyl group at the acyclic part, with long conjugated double bonds, only synthesized in fungi called red (oleaginous) yeasts, e.g., the genera Rhodotorula and Sporobolomyces. This unique red pigment with strong antioxidant properties is promising for use in food additives, nutritional supplements, and cosmetics. We aimed to produce torularhodin in Escherichia coli through the identification of the biosynthesis genes needed for its heterologous production, while no genes oxidizing torulene to torularhodin had been reported. The Rhodotorula toruloides crtI (CAR1) and crtYB (CAR2) genes, which were chemically synthesized, proved to lead to the complete conversion of phytoene into torulene when they were introduced into an E. coli cell that carried the Pantoea ananatis crtE and Haematococcus pluvialis IDI genes. We found that the Planococcus maritimus genes coding for C30 carotenoid terminal oxidase (crtP/crtNb/cruO) and aldehyde dehydrogenase (aldH/crtNc), through their introduction into the E. coli transformant synthesizing torulene, mediated the efficient oxidations of torulene to torularhodin, and resulted in the production of torularhodin as the dominant carotenoid. This is the first report of torularhodin production in a heterologous host. We also identified the aldH/crtNc gene in R. toruloides.

BioTech doi: 10.3390/biotech15010002

Authors: Sarah Döring Birte S. Wulfes Aleksandra Atanasova Carsten Jaeger Leopold Walzel Georg Tscheuschner Sabine Flemig Kornelia Gawlitza Ines Feldmann Zoltán Konthur Michael G. Weller

Reusable enzyme carriers are valuable for proteomic workflows, yet many supports are expensive or lack robustness. This study describes the covalent immobilization of recombinant trypsin on micrometer-sized corundum particles and assesses their performance in protein digestion and antibody analysis. The corundum surface was cleaned with potassium hydroxide, silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde. Recombinant trypsin was then attached, and the resulting imines were reduced with sodium cyanoborohydride. Aromatic amino acid analysis (AAAA) estimated an enzyme loading of approximately 1 µg/mg. Non-specific adsorption of human plasma proteins was suppressed by blocking residual aldehydes with a Tris-glycine-lysine buffer. Compared with free trypsin, immobilization shifted the temperature optimum from 50 to 60 °C and greatly improved stability in 1 M guanidinium hydrochloride. Activity remained above 80% across several reuse cycles, and storage at 4 °C preserved functionality for weeks. When applied to digesting the NISTmAb, immobilized trypsin provided peptide yields and sequence coverage comparable to soluble enzyme and outperformed it at elevated temperatures. MALDI-TOF MS analysis of Herceptin digests yielded fingerprint spectra that correctly identified the antibody and achieved >60% sequence coverage. The combination of low cost, robustness and analytical performance makes corundum-immobilized trypsin an attractive option for research and routine proteomic workflows.

BioTech doi: 10.3390/biotech15010001

Authors: Shabnam Ghanbarzadeh Yi Yuan Ehssan H. Koupaie

The transition toward a circular bioeconomy requires efficient conversion of biogenic wastes and biomass into renewable fuels. This study explores the gasification potential of wastewater sludge (WWS) and food waste (FW), representing high moisture-content biowastes, compared with softwood (SW), a lignocellulosic biomass reference. An Aspen Plus equilibrium model incorporating the drying stage was developed to evaluate the performance of air and steam gasification. The effects of temperature (400–1200 °C), equivalence ratio (ER = 0.1–1), and steam-to-biomass ratio (S/B = 0.1–1) on gas composition and energy efficiency (EE) were examined. Increasing temperature enhanced H2 and CO generation but reduced CH4, resulting in a maximum EE at intermediate temperatures, after which it declined due to the lower heating value of the gases. Although EE followed the order SW > FW > WWS, both biowastes maintained robust efficiencies (60–80%) despite high drying energy requirements. Steam gasification increased H2 content up to 53% (WWS), 54% (FW), and 51% (SW) near S/B = 0.5–0.6, while air gasification achieved 23–27% H2 and 70–80% EE at ER ≈ 0.1–0.2. The results confirm that wet bio-wastes such as WWS and FW can achieve performance comparable to lignocellulosic biomass, highlighting their suitability as sustainable feedstocks for waste-to-syngas conversion and supporting bioenergy integration into waste management systems.

BioTech doi: 10.3390/biotech14040099

Authors: Fanglian Lu Deqin Luo Lian Yang Ranran Dong

Nonylphenol (NP) bioremediation is constrained by the scarcity of efficient and non-pathogenic degrading strains. To clarify the role of acetyl-CoA C-acetyltransferase (AtoB) in NP degradation, we generated an atoB-overexpressed strain (LY-OE) from the environmentally tolerant Bacillus cereus LY and compared its degradation rate with the wild type using HPLC. Untargeted lipidomics was conducted to characterize metabolic responses under NP stress, and key differential lipid metabolites (DELMs) were further validated by ELISA. Additionally, AtoB concentration and ATP content were quantified using commercial assay kits in Bacillus cereus. LY-OE showed a markedly higher NP degradation rate (96%) than LY (85%). Lipidomic analysis identified 34 significant DELMs (VIP > 1, p < 0.05), including elevated cardiolipin (CL) and phosphatidylglycerol (PG), and reduced phosphatidylcholine (PC) and triglycerides (TG). ELISA confirmed these changes (p < 0.01 or p < 0.001), consistent with lipidomic findings. LY-OE showed significantly higher AtoB concentration during the logarithmic growth phase and exhibited higher ATP content during NP degradation. These findings suggest that atoB overexpression enhances NP degradation by both boosting energy supply and remodeling lipid metabolism. This work identifies atoB as a key factor for NP biodegradation and provides a promising strategy for developing high-performance bioremediation strains.

BioTech doi: 10.3390/biotech14040098

Authors: Mengya Liu Chi Zhang Lili Xu Md. Muedur Rahman Shoshiro Hirayama Shuhei Aramaki Atsushi Baba Ryo Omagari Yutaka Takahashi Tomoaki Kahyo Mitsutoshi Setou

Mass spectrometry imaging (MSI) visualizes the spatial distribution of biomolecules in tissues, whereas ion mobility–mass spectrometry (IM-MS) separates ions through the collision cross-section (CCS) with an inert gas, providing the structural characteristics of isomers. Recent advances have established an integrated workflow, ion mobility–mass spectrometry imaging (IM-MSI), that couples IM with MSI, uniting molecular discrimination with spatial mapping. This synergy has been widely applied in oncology and neuropsychiatric disorders, offering unprecedented insights into biomarker discovery and disease mechanisms. Here, we summarize the principles and classifications of IM-MSI, review their combined biomedical applications, and discuss data processing workflows and commonly used tools.

BioTech doi: 10.3390/biotech14040097

Authors: Cristián Raziel Delgado-González Ashutosh Sharma Margarita Islas-Pelcastre Mariana Saucedo-García Eliazar Aquino-Torres Jaime Pacheco-Trejo Silvia Armenta-Jaime Nallely Rivero-Pérez Alfredo Madariaga-Navarrete

The genus Kalanchoe (Crassulaceae) comprises approximately 125 species of succulents distributed across Madagascar, Africa, Arabia, Australia, Southeast Asia, and tropical America. Traditionally regarded as “miracle plants”, Kalanchoe species are employed for treating inflammatory, infectious, metabolic, and cardiovascular conditions; this is associated with their abundant content of polyphenols, including phenolic acids and flavonoids such as quercetin, kaempferol, luteolin, rutin, and patuletin. However, robust clinical evidence remains limited. This review integrates pharmacological and bioinformatic perspectives by analyzing more than 70 studies published since 2000 on 15 species, including Bryophyllum. As an in silico complement, the genome of Kalanchoe fedtschenkoi was used to predict genes (AUGUSTUS), perform homology searches against Arabidopsis thaliana, and model three key enzymes: CHS, CYP90, and VEP1. The AlphaFold2/ColabFold models showed conserved catalytic motifs, and molecular docking with representative ligands supported the plausibility of biosynthetic pathways for flavonoids, brassinosteroids, and bufadienolides. The available evidence highlights chemopreventive, antibacterial, anti-inflammatory, antiviral, antioxidant, and cytotoxic activities, primarily associated with flavonoids and bufadienolides. Significant gaps remain, such as the lack of gene–metabolite correlations and the absence of standardized clinical trials. Overall, Kalanchoe represents a promising model that requires multi-omics approaches to enhance its phytopharmaceutical potential.

BioTech doi: 10.3390/biotech14040096

Authors: Khin Than Yee Jason Macrander Olga Vasieva Ponlapat Rojnuckarin

In Myanmar, Russell’s viper (Daboia siamensis) bite is a significant public health problem. In this study, we expend upon our previous RNA-sequencing approach to characterize candidate toxin genes encoding D. siamensis toxins. The mRNA was extracted from Myanmar Russell’s viper venom glands. The RNAseq was performed using Illumina next-generation sequencing. Subsequently, candidate toxin transcripts were recognized by the Venomix pipeline. This study focused on 29 unique cDNA sequences representing eight newly identified venom gene families with low-to-moderate expression levels. These transcripts represented 0.088% of the total number of transcripts in the dataset. The translated protein sequences were analyzed for their conserved motifs and domains to predict their functions. They were neprilysins (bioactive peptide inactivators), cystatins (protease inhibitors with anti-metastatic activities), waprin and vipericidin (antimicrobial peptides), veficolin (platelet and complement activation), vespryns and three-finger toxins (elapid toxin homologs causing neurotoxic activity and tissue damage), and endothelial lipases (unknown function). Their functional activities should be further investigated for potential therapeutic applications, for example, in cancer or antibiotic-resistant infections.

BioTech doi: 10.3390/biotech14040095

Authors: Ximena Vázquez-Cadena Oscar Alejandro Faz-Cortez Benito Pereyra-Alférez César Ignacio Hernández-Vásquez Luis Jesús Galán-Wong Myriam Elías-Santos Jorge Hugo Garcia-Garcia

MicroRNAs (miRNAs) are small single-stranded non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Recent studies have demonstrated that plant miRNAs can survive through dietary intake and act as signaling molecules in intercellular communication, proving a cross-kingdom interaction. The aim of the present study was to use computational approaches to identify interactions between Zea mays (maize) miRNAs and human coding mRNAs potentially involved in different biological processes. We identified 961 unique genes potentially regulated by maize miRNAs. Furthermore, functional enrichment analysis via GO and KEGG was carried out focusing primarily on the pathway related to prostate cancer where 13 genes were potentially regulated by 15 maize miRNAs. Our findings not only provide an important insight into the potential effects that maize-derived miRNAs could have on the human body, but also highlight the importance of considering these molecules for further research and potential therapeutic applications against major diseases such as cancer.

BioTech doi: 10.3390/biotech14040094

Authors: Srichandan Padhi Swati Sharma Puja Sarkar Marco Masi Alessio Cimmino Amit Kumar Rai

Helicobacter pylori, the gastric pathogen which colonizes the gastric mucosa of more than half of the world’s population, is considered a risk factor for peptic ulcers and is epidemiologically associated with gastric cancer. Antimicrobial eradication of this pathogen has now become a central concern because of its growing resistance to frontline antibiotics such as clarithromycin and metronidazole. Moreover, these antibiotics can have adverse effects on the normal human gut flora and can lead to several health complications. Most times, the antibiotic doses become intolerable to the elderly population and they reject the therapy. This has impelled us to think about alternate effective and safe antimicrobials which can replace antibiotic usage or may reduce their dosage when used together with the antibiotics. Plant and microbial natural products, in view of this, offer an excellent source of novel and potential antimicrobial agents. Herein, we review anti-H. pylori natural compounds from diverse plant and microbial sources and highlight their role in the management of H. pylori infection.

BioTech doi: 10.3390/biotech14040093

Authors: Marco A. Ramírez-Mosqueda Jorge David Cadena-Zamudio Carlos A. Cruz-Cruz José Luis Aguirre-Noyola Raúl Barbón Rafael Gómez-Kosky Carlos Angulo

Somatic embryogenesis (SE) is a morphogenetic pathway widely employed in the commercial micropropagation of plants. This route enables the generation of somatic embryos from somatic tissues, which give rise to complete (bipolar) plants that develop like zygotic embryos. SE can proceed via direct or indirect pathways, and both approaches have been adapted not only for large-scale clonal propagation but also for the regeneration of genetically modified plants. In this context, SE can be harnessed as a versatile platform for recombinant protein production, including vaccine antigens and therapeutic proteins, by combining plant tissue culture with genetic transformation strategies. Successful examples include non-model plants, as Daucus carota and Eleutherococcus senticosus expressing the cholera and heat-labile enterotoxin B subunits, respectively; Oryza sativa, Nicotiana tabacum, and Medicago sativa producing complex proteins such as human serum albumin (HSA), α1-antitrypsin (AAT), and monoclonal antibodies. However, challenges remain in optimizing transformation efficiency, scaling up bioreactor-based suspension cultures, and ensuring proper post-translational modifications under Good Manufacturing Practice (GMP) standards. Recent advances in synthetic biology, modular vector design, and glycoengineering have begun to address these limitations, improving control over transcriptional regulation and protein quality. This review highlights the application of SE as a biotechnological route for recombinant protein production, discusses current challenges, and presents innovative strategies and perspectives for the development of sustainable plant-derived biopharmaceutical systems.

BioTech doi: 10.3390/biotech14040092

Authors: Shuqi Chen Haoran Shi Peng Zhao Zengqiang Ma Xiaolong Li Xiangyu Wang Feiyan Xue

Background: Sunflower meal (SFM), a promising feed material, is constrained by its high content of crude fiber (CF) and chlorogenic acid (CGA). Methods: This study utilized a synergistic solid-state fermentation process involving the Bacillus subtilis strain B3 and the enzyme β-glucanase to enhance SFM’s application potential. Results: The synergistic treatment notably reduced CF by 12.7% and CGA by 99.77%, while simultaneously increasing acid-soluble protein and reducing sugar by 111.3% and 283.1%, respectively. Positive impacts on its physical structure, characterized by a looser network with visible pores, and on its microbial community, evidenced by an enriched abundance of fungal species such as Cyberlindnera and Aspergillus, were also observed. In vitro assays indicated improved digestibility of dry matter, neutral detergent fiber, and crude protein, along with a non-significant reduction in methane production. Conclusions: These results demonstrate that microbial-enzymatic synergy effectively enhances SFM’s nutritional profile.

BioTech doi: 10.3390/biotech14040091

Authors: Marcela Blažková Ľubica Uváčková Mária Maliarová Jozef Sokol Jana Viskupičová Tibor Maliar

Oxidative stress reflects an imbalance between pro-oxidants and antioxidants arising from physiological or environmental factors. Here, we applied our previously developed in situ microplate method for the simultaneous determination of antioxidant and pro-oxidant activities to compounds produced by plant cell cultures in vitro. The primary aim was to evaluate the added value of these compounds, which are widely used as additives in food, cosmetic, and pharmaceutical products. The secondary aim was to assess whether a predominance of pro-oxidant activity could limit their biotechnological production. Thirty-three compounds known to be produced by in vitro cultures (polyphenolic acids, flavonoids, quinones, alkaloids, etc.) were tested, and the pro-oxidant–antioxidant balance index (PABI) was calculated. Sixteen compounds showed measurable activities with DPPH50/FRAP50 values below 2 mM. Within this set, rosmarinic acid exhibited pronounced pro-oxidant behavior, whereas gallic acid, chlorogenic acid, and the anthocyanin cyanidin showed higher antioxidant potency and favorable PABI values. Such compounds may deliver added benefits when incorporated into food or cosmetic products and are unlikely to limit production in cell culture.

BioTech doi: 10.3390/biotech14040090

Authors: Anastasia D. Novokshonova Pavel V. Khramtsov Maksim V. Dmitriev Ekaterina E. Khramtsova

Acylpyruvate derivatives represent a promising yet underexplored class of compounds for modulating microalgal growth and metabolism. Inspired by the metabolic role of pyruvate and the diverse bioactivity of its acylated analogs, this study investigates the structure–activity relationship of a diverse library of 55 acylpyruvate-derived compounds for stimulation of the green microalga Chlorella vulgaris. The library, encompassing 12 chemotypes including acylpyruvic acids, their esters, and various heterocyclic derivatives, was screened for effects on C. vulgaris growth. Six compounds were identified as active ones that enhanced biomass production in a preliminary microassay. Notably, four of these active compounds were direct acylpyruvate derivatives, highlighting this scaffold as the most promising one. Conversely, a specific subclass, 1,4-benzoxazin-2-ones, exhibited potent, dose-dependent algicidal activity. Detailed assessment of the active compounds under scaled-up culture conditions revealed that while their effect on overall cell density was limited, several compounds significantly enhanced the intracellular content of valuable metabolites: one increased chlorophyll content by 17%, another elevated carotenoids by 40%, and a third boosted neutral lipid accumulation by 44%. Furthermore, control experiments confirmed that the bioactivity of p-ethoxybenzoylpyruvates, which showed the best biological activity, is inherent in the intact framework and is not mediated by their hydrolysis products. Our findings underscore the potential of acylpyruvates as versatile tools for the enhancement of metabolite production in microalgae and as potent candidates for the development of algicides.

BioTech doi: 10.3390/biotech14040089

Authors: Georgios Mitronikas Athina Voudanta Aliki Kapazoglou Maria Gerakari Eleni M. Abraham Eleni Tani Vasileios Papasotiropoulos

The growing demand for sustainable, health-promoting foods has intensified efforts to improve the antioxidant potential of berry crops through integrative agronomic, genomic, and breeding innovations. Berries are rich dietary sources of bioactive compounds that support human health and provide benefits far beyond basic nutrition. This review explores the diversity of major berry crops, including blueberries, raspberries, cranberries, blackberries, and grapes, with emphasis on their nutritional value and antioxidant profiles. It also examines their domestication history, wild relatives, and commercial cultivars, offering insight into the genetic and phenotypic diversity underlying their rich chemical composition. Furthermore, the review highlights the application of modern tools to enhance antioxidant content. By integrating agronomic practices such as seed priming and grafting, advanced molecular breeding technologies, including multi-omics, genome-wide association studies (GWAS), and genome editing, breeders and researchers can accelerate the development of high-value berry cultivars that combine superior nutritional quality, resilience to environmental stress, and sustainable productivity under the challenges posed by climate change.

BioTech doi: 10.3390/biotech14040088

Authors: Emi Inada Issei Saitoh Masahiko Terajima Yuki Kiyokawa Naoko Kubota Haruyoshi Yamaza Kazunori Morohoshi Shingo Nakamura Masahiro Sato

The fields of regenerative medicine and stem cell-based tissue engineering hold great potential for treating a wide range of tissue and organ defects. Stem cells are ideal candidates for regenerative medicine because they are undifferentiated cells with the capacity for self-renewal, rapid proliferation, multilineage differentiation, and expression of pluripotency-associated genes. Human dental pulp stem cells (DPSCs) consist of various cell types (including stem cells) and possess multilineage differentiation potential. Owing to their easy isolation and rapid proliferation, DPSCs and their derivatives are considered promising candidates for repairing injured tissues. Recent advances in gene engineering have enabled cells to express specific genes of interest, leading to the secretion of medically important proteins or the alteration of cell behavior. For example, transient expression of Yamanaka’s factors in DPSCs can induce transdifferentiation into induced pluripotent stem cells (iPSCs). These gene-engineered cells represent valuable candidates for regenerative medicine, including stem cell therapies and tissue engineering. However, challenges remain in their development and application, particularly regarding safety, efficacy, and scalability. This review summarizes current knowledge on gene-engineered DPSCs and their derivatives and explores possible clinical applications, with a special focus on oral regeneration.

BioTech doi: 10.3390/biotech14040087

Authors: Anh T. Tran Junhao Wen Gaby Abou Karam Dorin Zeevi Adnan I. Qureshi Ajay Malhotra Shahram Majidi Niloufar Valizadeh Santosh B. Murthy Mert R. Sabuncu David Roh Guido J. Falcone Kevin N. Sheth Seyedmehdi Payabvash

Handcrafted radiomics use predefined formulas to extract quantitative features from medical images, whereas deep neural networks learn de novo features through iterative training. We compared these approaches for predicting 3-month outcomes and hematoma expansion from admission non-contrast head CT in acute intracerebral hemorrhage (ICH). Training and cross-validation were performed using a multicenter trial cohort (n = 866), with external validation on a single-center dataset (n = 645). We trained multiscale U-shaped segmentation models for hematoma segmentation and extracted (i) radiomics from the segmented lesions and (ii) two latent deep feature sets—from the segmentation encoder and a generative autoencoder trained on dilated lesion patches. Features were reduced with unsupervised Non-Negative Matrix Factorization (NMF) to 128 per set and used—alone or in combination—for six machine-learning classifiers to predict 3-month clinical outcomes and (>3, >6, >9 mL) hematoma expansion thresholds. The addition of latent deep features to radiomics numerically increased model prediction performance for 3-month outcomes and hematoma expansion using Random Forest, XGBoost, Extra Trees, or Elastic Net classifiers; however, the improved accuracy only reached statistical significance in predicting >3 mL hematoma expansion. Clinically, these consistent but modest increases in prediction performance may improve risk stratification at the individual level. Nevertheless, the latent deep features show potential for extracting additional clinically relevant information from admission head CT for prognostication in hemorrhagic stroke.

BioTech doi: 10.3390/biotech14040086

Authors: Laura Martínez-Alarcón Sergio Liarte Juana M. Abellaneda Juan J. Quereda Livia Mendonça Antonio Muñoz Pablo Ramírez Guillermo Ramis

Cell transplantation is often performed with ultrasonographic guidance for accurate delivery through injection. In such procedures, using ultrasonographic contrast greatly improves target delivery. However, accumulating evidence suggests that exposure to such contrast agents may have negative effects on transplanted cells. No study so far has researched this issue. Stabilized sulfur hexafluoride (SF6) microbubbles are a widely used sonographic contrast agent. Skin hCD55 porcine transgenic fibroblasts and mesenchymal stem cells from human bone marrow (hMSCs) were exposed in vitro to SF6 in concentrations ranging from 1.54 µM to 308 µM. The effects on viability and cell growth were registered using an impedance-based label-free Real-Time Cell Analyzer (RTCA). Data was recorded every 15 min for 50 h of total study time. Both cell lines behave distinctly when exposed to SF6. Porcine fibroblast growth showed relevant alterations only when exposed to higher concentrations. In contrast, hMSCs showed progressive growth decrease in relation to SF6 concentration. Taken together, while SF6-based contrast agents pose no threat to patient safety, our results indicate that exposure of suspended stem cells to the contrast agent could affect the effective dose administered in cell therapy procedures. This prompts specific cell lineage testing, adjusting methods and properly compensating for cell loss, with a potential impact on procedural cost and success rates.

BioTech doi: 10.3390/biotech14040085

Authors: Afifa Husna Agustin Krisna Wardani Chun-Yi Hu Yo-Chia Chen

Xylooligosaccharides (XOS) are functional oligosaccharides with recognized prebiotic properties and growing industrial relevance, typically obtained through enzymatic depolymerization of xylan-rich lignocellulosic substrates. In this study, a recombinant endo-β-1,4-xylanase (XynA) from Clostridium acetobutylicum was employed for XOS production. The xynA gene was cloned into the expression vector pET-21a(+) and heterologously expressed in Escherichia coli BL21(DE3) under induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein, with an estimated molecular mass of 37.5 kDa, was verified by SDS-PAGE and Western blot analysis. Functional characterization via thin-layer chromatography revealed that XynA efficiently hydrolyzed beechwood xylan and rye arabinoxylan, predominantly yielding xylobiose. Additionally, the enzyme catalyzed the conversion of xylotriose into xylobiose and trace amounts of xylose. Notably, XynA demonstrated hydrolytic activity against autohydrolysed and alkali-pretreated coconut husk biomass, facilitating the release of XOS. These results underscore the potential of C. acetobutylicum XynA as a biocatalyst for the valorization of lignocellulosic residues into high-value oligosaccharides.

BioTech doi: 10.3390/biotech14040084

Authors: Julian Tix Fernando Pedraza Roland Ulber Nils Tippkötter

Carbon capture and power-to-X are becoming increasingly relevant in the context of decarbonization and supply security. Actinobacillus succinogenes is capable of transforming CO2 into succinate, whereby product formation is significantly limited by the availability of NADH. The aim of this work was to further develop a bioelectrochemical system (BES) in order to provide additional reduction equivalents and thus increase yield and titer. To this end, a new BES configuration was established. A conventional stirred tank reactor (STR) is coupled via a bypass to an H-cell, in which the redox mediator neutral red (NR) is electrochemically reduced and then returned back to the bioreactor. The indirect electron transfer decouples the electrochemical reduction from the biology and results in increased intracellular availability of NADH. The present approach resulted in an increase in yield from 0.64 g·g−1 to 0.76 g·g−1, corresponding to an increase of 18%. At the same time, a titer of 16.48 ± 0.19 g·L−1 was achieved in the BES, compared to 12.05 ± 0.18 g·L−1 in the control. The results show that the mediator-assisted, partially decoupled BES architecture significantly improves CO2-based succinate production and opens up a scalable path to the use of renewable electricity as a reduction source in power-to-X processes.

BioTech doi: 10.3390/biotech14040083

Authors: Aoqi Dong Xiaoying Dong Xinying Dai Yanru Gao Yuewen Ning Xiya Fan Haiyan Liu

Suaeda salsa, an annual herb belonging to the genus Suaeda within the Chenopodiaceae family, is highly salt-tolerant and can thrive in large quantities on saline and alkaline soils. This study presents a novel fermentation technique to produce Suaeda tea, utilizing a synergistic blend of microbial agents: Kluyveromyces marxianus, Komagataeibacter europaeus, and Acetobacter schutzenbachii. The resulting tea demonstrates a potent antioxidant capacity, with a hydroxyl radical scavenging rate of 64.2% and an exceptional 1,1-diphenyl-2-picrylhydrazyl radical scavenging capacity of 83.3%, along with increased ferric ion reduction/antioxidant power (FRAP) reducing power (1.82), indicating its superior antioxidant profile. Through the comparison of different microbial strain combinations under varying process parameters such as fermentation temperature and duration, the experiment revealed that fermentation at 37 °C for 24 h results in the highest concentrations of tea polyphenols (TPs) (≥10.87 mg/mL) and free amino acids (26.32 mg/100 mL). The quality of the fermented Suaeda tea meets the stringent GB/T 21733-2008 standards for tea beverages, exhibiting excellent physicochemical indices and sensory attributes. The antioxidant efficacy of the fermented Suaeda tea persists significantly throughout a 180-day duration. The optimization of the fermentation process for Suaeda tea not only provides a theoretical framework for large-scale production but also establishes a foundation for Suaeda salsa in the tea beverage sector. This innovation enriches the market with a diverse range of health-promoting teas, catering to the growing consumer demand for nutritious and beneficial beverages.

BioTech doi: 10.3390/biotech14040082

Authors: Iridiam Hernández-Soto Antonio Juárez-Maldonado Alfredo Madariaga-Navarrete Ashutosh Sharma Antonio de Jesus Cenobio-Galindo Jose Manuel Pinedo-Espinoza Aracely Hernández-Pérez Alma Delia Hernández-Fuentes

Argemone mexicana L. is considered a weed; however, it contains secondary metabolites that can control phytopathogenic fungi in vitro, with the potential to adapt its effectiveness in the field. In the present study, leaf extracts of A. mexicana (hexane and methanol) were prepared, and their chemical profiles were analyzed using gas chromatography–mass spectrometry (GC-MS). The in vitro antifungal activity of each extract was evaluated at different concentrations (500, 1000, 2000, 4000, and 8000 mg L−1) against phytopathogens such as Monilinia fructicola, Colletotrichum gloeosporioides, Fusarium oxysporum, and Sclerotinia sclerotiorum. Based on their chemical profiles, 14 compounds were identified in the hexanic extract, and 11 compounds were identified in the methanolic extract. These compounds included those with antifungal activity, such as Benzene; 1.3-bis(1.1-dimethylethyl)-; pentanoic acid; 5-hydroxy-, 2,4-di-1-butylphenyl esters; 1,2,4-Triazol-4-amine; and N-(2-thienylmethyl). The hexanic extract demonstrated fungistatic activity on the four fungi tested, while the methanolic extract exhibited fungicidal activity against C. gloeosporioides and F. oxysporum. The results of the Probit analysis showed variations in the sensitivity of phytopathogenic fungi to the treatments evaluated. In M. fructicola, the hexane extract presented an EC50 of 317,146 mg L−1 and an EC90 of 400,796 mg L−1. For C. gloeosporioides, the EC50 was 2676 mg L−1 and the EC90 was 888,177 mg L−1, while in F. oxysporum an EC50 of 34,274 mg L−1 and an EC90 of 1528 mg L−1 were estimated. In the case of S. sclerotiorum, an EC50 of 560 mg L−1 and an EC90 of 7776 mg L−1 were obtained. Finally, for the commercial fungicide Captan®, an EC50 of 1.19 mg L−1 and an EC90 of 1.67 mg L−1. These results suggest that extracts from A. mexicana could provide a natural alternative for the control of phytopathogenic fungi.

BioTech doi: 10.3390/biotech14040081

Authors: Kazunori Morohoshi Miho Ohba Masahiro Sato Shingo Nakamura

Newborn mice (up to 6 d after birth) are suitable for genetic manipulations, such as facial vein-mediated injection, owing to their hairless and thin skin. Their small body volumes also facilitate the rapid dissemination of injected solutions, supporting gene engineering-related experiments. However, anesthesia in newborns is challenging because of the potential risks associated with anesthetic agents. Isoflurane inhalation anesthesia is an option, although its effects on brain development remain under investigation. In this study, we established a reproducible protocol for delivering nucleic acids to juvenile mouse testes using a simple isoflurane-based anesthetic system prepared from common laboratory equipment. Using this system, nucleic acids were successfully delivered to juvenile mouse testes via intra-testicular injection, followed by in vivo electroporation. The present isoflurane-based method achieved >90% postoperative survival with normal maternal nursing observations. Gene delivery resulted in limited transfection of seminiferous tubules but efficient interstitial Leydig cell transfection. Thus, gene engineering in somatic and germ cells in neonatal mice will be facilitated using the anesthetic protocol established in this study.

BioTech doi: 10.3390/biotech14040080

Authors: María Isabel Iñiguez-Luna Jorge David Cadena-Zamudio Marco A. Ramírez-Mosqueda José Luis Aguirre-Noyola Daniel Alejandro Cadena-Zamudio Jorge Cadena-Iñiguez Alma Armenta-Medina

Molecular docking has emerged as a pivotal computational approach in agri-food research, offering a rapid and targeted means to discover bioactive molecules for crop protection and food safety. Its ability to predict and visualize interactions between natural or synthetic compounds and specific biological targets provides valuable opportunities to address urgent agricultural challenges, including climate change and the rise in resistant crop pathogens. By enabling the in silico screening of diverse chemical entities, this technique facilitates the identification of molecules with antimicrobial and antifungal properties, specifically designed to interact with critical enzymatic pathways in plant pathogens. Recent advancements, such as the integration of molecular dynamics simulations and artificial intelligence-enhanced scoring functions, have significantly improved docking accuracy by addressing limitations like protein flexibility and solvent effects. These technological improvements have accelerated the discovery of eco-friendly biopesticides and multifunctional nutraceutical agents. Promising developments include nanoparticle-based delivery systems that enhance the stability and efficacy of bioactive molecules. Despite its potential, molecular docking still faces challenges related to incomplete protein structures, variability in scoring algorithms, and limited experimental validation in agricultural contexts. This work highlights these limitations while outlining current trends and future prospects to guide its effective application in agri-food biotechnology.

BioTech doi: 10.3390/biotech14040079

Authors: Gloria Sciuto Nunziatina Russo Cinzia L. Randazzo Cinzia Caggia

The dairy sector produces considerable amounts of nutrient-rich effluents, which are frequently undervalued as simple by-products or waste. In particular, Second Cheese Whey (SCW), also known as scotta, exhausted whey, or deproteinized whey, represents the liquid fraction from ricotta cheese production. Despite its abundance and high organic and saline content, SCW is often improperly discharged into terrestrial and aquatic ecosystems, causing both environmental impact and resource waste. The available purification methods are expensive for dairy companies, and, at best, SCW is reused as feed or fertilizer. In recent years, increasing awareness of sustainability and circular economy principles has increased interest in the valorization of SCW. Biological treatment of SCW using microalgae represents an attractive strategy, as it simultaneously reduces the organic load and converts waste into algal biomass. This biomass can be further valorized as a source of proteins, pigments, and bioactive compounds with industrial relevance, supporting applications in food, nutraceuticals, biofuels, and cosmetics. This review, starting from analyzing the characteristics, production volumes, and environmental issues associated with SCW, focused on the potential of microalgae application for their valorization. In addition, the broader regulatory and sustainability aspects related to biomass utilization and treated SCW are considered, highlighting both the promises and limitations of microalgae-based strategies by integrating technological prospects with policy considerations.

BioTech doi: 10.3390/biotech14040078

Authors: Tata Ninidze Tamar Koberidze Kakha Bitskinashvili Tamara Kutateladze Boris Vishnepolsky Nelly Datukishvili

The detection of allergens is essential for ensuring food safety, protecting public health, and providing accurate information to consumers. Wheat (Triticum aestivum L.) and maize (Zea mays L.) are recognized as important food allergens. In this study, novel PCR methods were developed for the reliable detection of wheat and maize allergens, including wheat high-molecular-weight glutenin subunit (HMW-GS) and low-molecular-weight glutenin subunit (LMW-GS), as well as three maize allergens, namely, Zea m 14, Zea m 8, and zein. Wheat and maize genomic DNA, as well as allergen genes, were examined during 60 min of baking at 180 °C and 220 °C. Agarose gel electrophoresis revealed degradation of genomic DNA and amplified PCR fragments in correlation with increasing baking temperature and time. For each target gene, the best primers were identified that could detect HMW-GS and LMW-GS genes in wheat samples and Zea m 14, Zea m 8, and zein genes in maize samples after baking at 220 °C for 60 min and 40 min, respectively. The results indicate that these PCR methods can be used for the reliable and sensitive detection of wheat and maize allergens in processed foods.

BioTech doi: 10.3390/biotech14040077

Authors: Mario James-Forest Ma del Carmen Ojeda-Zacarías Alhagie K. Cham Héctor Lozoya-Saldaña Rigoberto E. Vázquez-Alvarado Emilio Olivares-Sáenz Alejandro Ibarra-López

This study evaluates the impact of biotic elicitors and hormone regimes on the in vitro establishment, shoot multiplication, and organogenesis of Solanum tuberosum L. cv. Fianna under controlled laboratory conditions. Explants derived from pre-treated tubers were cultured on Murashige and Skoog (MS) medium supplemented with vitamins and varying concentrations of growth regulators or elicitors. Aseptic establishment achieved a high success rate (~95%) using a 6% sodium hypochlorite disinfection protocol. Multiplication was significantly enhanced with a combination of 0.2 mg L−1 naphthaleneacetic acid (NAA) and 0.5–1.0 mg L−1 benzylaminopurine (BAP), producing the greatest number and length of shoots and roots. Direct organogenesis was stimulated by bio-elicitors Activane®, Micobiol®, and Stemicol® in (MS) basal medium at mid-level concentrations (0.5 g or mL L−1), improving shoot number, elongation, and root development. Activane®, Micobiol®, and Stemicol® are commercial elicitors that stimulate plant defense pathways and morphogenesis through salicylic acid, microbial, and jasmonic acid signaling mechanisms, respectively. Indirect organogenesis showed significantly higher callus proliferation in Stemicol® and Micobiol® treatments compared to the control medium, resulting in the highest fresh weight, diameter, and friability of callus. The results demonstrate the potential of biotic elicitors as alternatives or enhancers to traditional plant growth regulators in potato tissue culture, supporting more efficient and cost-effective micropropagation strategies.

BioTech doi: 10.3390/biotech14040076

Authors: Taner Sar Clarisse Uwineza Mohammad J. Taherzadeh Amir Mahboubi

Organic-waste-derived volatile fatty acids (VFAs) are promising substrates for fungal biomass cultivation, offering a nutrient-rich medium capable of meeting microbial growth requirements. However, the growth and biomass productivity are highly influenced by the VFAs’ composition and mode of operation. This study investigated the cultivation of Aspergillus oryzae fungal biomass using agro-industrial-derived VFA effluent, employing repeated-batch and fed-batch (stepwise and continuous-feeding) cultivation modes to evaluate fungal growth and biomass composition. The highest dry biomass yield of 0.41 dry biomass/gVFAsfed (g/g) was achieved in fed-batch mode with continuous feeding, where the biomass exhibited pellet morphology, facilitating ease of harvesting. The crude protein content varied according to the cultivation strategy, reaching 45–53% in continuous-feeding fed-batch mode, while it was 34–42% in stepwise fed-batch mode. Additionally, the fungal biomass contained significant levels of essential macronutrients and trace elements, including Mg, Ca, K, Mn, and Fe, which are crucial if the biomass is intended to be used in animal feed formulations. This study highlights the effects of cultivation modes on biomass composition and the potential of VFA-derived fungal biomass as a sustainable feed ingredient.

BioTech doi: 10.3390/biotech14030075

Authors: Ghazaleh Khalili-Tanha Alireza Shoari

Breast cancer is the most prevalent cancer among women and is challenging to diagnose and treat due to its diverse subtypes and stages. Precision medicine aims to improve early detection, prognosis, and treatment planning by identifying new clinical biomarkers. The review emphasizes the importance of using cutting-edge technology and artificial intelligence (AI) to identify new biomarkers associated with epithelial–mesenchymal transition (EMT). During EMT, epithelial cells transform into a mesenchymal state, a process driven by genetic and epigenetic alterations that facilitate cancer progression. The review discusses how statistical analysis and machine learning methods applied to multi-omics data facilitate the discovery of novel EMT-related biomarkers, thereby advancing therapeutic strategies. This conclusion is supported by numerous clinical and preclinical studies on breast cancer.

BioTech doi: 10.3390/biotech14030074

Authors: Fatima Zohra Kaissar Khelifa Bouacem Mohammed Lamine Benine Sondes Mechri Shubha Rani Sharma Vishal Kumar Singh Mahfoud Bakli Seif El Islam Lebouachera Giovanni Emiliani

Pectins are high-value plant cell-wall polysaccharides with extensive applications in the food, pharmaceutical, textile, paper, and environmental sectors. Traditional extraction and processing methodologies rely heavily on harsh acids, high temperatures, and non-renewable solvents, generating substantial environmental and economic costs. This review consolidates recent advances across the entire Bacillus–pectinase value chain, from green pectin extraction and upstream substrate characterization, through process and statistical optimization of enzyme production, to industrial biocatalysis applications. We propose a practical roadmap for developing high-efficiency, low-environmental-footprint enzyme systems that support circular bioeconomy objectives. Critical evaluation of optimization strategies, including submerged versus solid-state fermentation, response surface methodology, artificial neural networks, and design of experiments, is supported by comparative data on strain performance, fermentation parameters, and industrial titers. Sector-specific case studies demonstrate the efficacy of Bacillus pectinases in fruit-juice clarification, textile bio-scouring, paper bio-bleaching, bio-based detergents, coffee and tea processing, oil extraction, animal feed enhancement, wastewater treatment, and plant-virus purification. Remaining challenges, including enzyme stability in complex matrices, techno-economic scale-up, and structure-guided protein engineering, are identified. Future directions are charted toward CRISPR-driven enzyme design and fully integrated circular-economy bioprocessing platforms.

BioTech doi: 10.3390/biotech14030073

Authors: Kisook Jung Ick-hyun Jo Bae Young Choi Jaewook Kim

Models that predict the 3D structure of proteins enable us to easily analyze the structure of unknown proteins. Though many of these models have been found to be accurate, their application in plant proteins is not always entirely accurate. Thus, we aimed to develop a versatile yet simple pipeline that can predict novel proteins with a specific function. As an example, via benchmark studies, we sought to discover novel UDP-glycosyltransferases (UGTs) potentially involved in ginsenoside biosynthesis. Since the functionality of these UGTs has been shown to be determined by a few amino acids, a 3D-structure-based pipeline was required. Our pipeline includes four sequential steps: a sequence-based homology search, AlphaFold3-based 3D structure prediction, docking simulations with ginsenoside intermediates using SwissDock and CB-Dock2, and MPEK analysis to assess interaction stability. Through the application of this benchmark, we optimized the role of each module in the pipeline and successfully identified four novel UGT candidates. These candidates are predicted to catalyze the conversion of protopanaxadiol (PPD) to compound K (CK) or protopanaxatriol (PPT) to ginsenoside F1. This pilot study demonstrates how our pipeline can be used for the functional annotation of plant proteins and the discovery of enzymes involved in specialized pathways.

BioTech doi: 10.3390/biotech14030072

Authors: Kabelo P. Mokgopa Shina D. Oloniiju Kevin A. Lobb Tendamudzimu Tshiwawa

While databases are emerging across various domains, from small molecules to genomics and proteins, aptamer databases remain scarce, if not entirely absent. Such databases could serve as a comprehensive resource for advancing research, innovation, and the applications of aptamer technology across multiple fields. This advancement would likely lead to improvements in healthcare, environmental monitoring, and biotechnology. Furthermore, the establishment of aptamer databases would facilitate molecular modelling and machine learning, opening doors to further advancements in understanding and utilizing aptamers. Against this backdrop, in this study, we present and benchmark the Base Randomization Algorithm (BRA) as a potential solution to the scarcity of aptamer databases. Through statistical analysis, we examine key factors such as minimum free energy (MFE), base compositions, and base arrangements. Notably, sequences generated using the BRA exhibit a Gaussian distribution pattern. We also examine the details of how each base within a sequence is chosen using mathematical principles, ensuring that the sequences are valid and optimized statistically. Additionally, we explore how the length of the randomized generated sequences can affect the folding of their structures at both the secondary and tertiary levels. Based on composition analysis, we propose that the base mean of the dataset can be approximated as x¯B≈Px × N, for dataset of sequences with the same length and x¯B≈Px × M,  where M  is the median and N the mean, for a dataset with randomized length that follows a Gaussian distribution.

BioTech doi: 10.3390/biotech14030071

Authors: Evgenia Rizou Nikolaos Monokrousos Triantafyllia Kardami Georgia V. Baliota Christos I. Rumbos Christos G. Athanassiou Nikolaos Tsiropoulos Nikoletta Ntalli

Insect-derived frass is gaining attention as a circular bioeconomy product with fertilizing and pest-suppressive potential. This study investigates Tenebrio molitor frass as a soil amendment for promoting beneficial nematodes and suppressing Meloidogyne incognita. A 40-day pot experiment on clay loam soil tested with six inputs: raw and heat-treated frass (0.5%, 1% w/w), Melia azedarach fruit powder (1.6%), and an untreated control. Soil nematode communities were assessed at 5 and 40 days after application (DAA), and nematicidal activity was evaluated in vitro. Raw frass at 1% induced a rapid response from free-living nematodes at 5 DAA, with increased abundance of bacterivorous taxa such as Rhabditis and Acrobeloides, alongside a higher Enrichment Index (EI), indicating short-term nutrient availability. At 40 DAA, only 1% raw frass consistently supported more cp-1 bacterivores and slightly increased Shannon diversity. Network analysis revealed more connected, modular structures in raw frass treatments, suggesting enhanced food web complexity. However, omnivore and predator effects were limited. Raw frass extracts caused over 80% paralysis of Meloidogyne incognita juveniles within 24 h, significantly outperforming heat-treated frass and Melia extracts. T. molitor frass moderately stimulates opportunistic nematodes and provides strong nematicidal effects, supporting its potential as a multifunctional input for sustainable soil management.

BioTech doi: 10.3390/biotech14030070

Authors: Pablo Ruiz-Amezcua Miguel Nieto Hernández Javier García Flores Clara Plaza Alonso David Reigada Teresa Muñoz-Galdeano Eva Vargas Rodrigo M. Maza Francisco J. Esteban Manuel Nieto-Díaz

The adult lumbar spinal cord plays a critical role in locomotor control and somatosensory integration, whose transcriptional architecture under physiological conditions has been characterized in various studies with restricted numbers of individuals (up to four). Here, we present an integrative single-nucleus RNA sequencing (snRNA-seq) atlas of the healthy adult mouse lumbar spinal cord, assembled from over 86,000 nuclei from 16 samples across five public datasets. Using a harmonized computational pipeline, we identify all major spinal cell lineages and resolve 17 transcriptionally distinct neuronal subtypes. A central novelty of our approach is the systematic inclusion of non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) and pseudogenes. By comparing transcriptomic analyses based on coding-only, non-coding-only, and combined gene sets, we show that ncRNAs, despite accounting to a 10% of the recorded information of each cell, contribute to cell type-specific signatures. This resource offers a high-resolution, ncRNA-inclusive reference for the adult spinal cord and provides a foundation for future studies on spinal plasticity, injury, and regeneration.

BioTech doi: 10.3390/biotech14030069

Authors: Eva Gómez-Molina Pedro Marco Sergi Garcia-Barreda Vicente González Sergio Sánchez

The success of truffle cultivation is especially dependent on the quality of truffle-mycorrhized seedlings, which are typically produced in nurseries under aseptic conditions to avoid root colonization by undesired ectomycorrhizal fungi. However, such practices may also eliminate beneficial microorganisms that could support truffle symbiosis and improve seedling quality. In this study, twelve endophytic bacterial and fungal strains, isolated from the Tuber melanosporum environment (gleba tissue, mycorrhizae and truffle brûlé), were tested for their effect on T. melanosporum mycorrhization levels in inoculated Quercus ilex seedlings under nursery conditions. Co-inoculation with a strain of Agrobacterium tumefaciens significantly enhanced root colonization by T. melanosporum, supporting its potential role as mycorrhizal helper bacterium. In contrast, a strain of Trichoderma harzianum negatively affected mycorrhization. The remaining strains did not show significant effects on seedling mycorrhization or seedling growth. Our findings support the hypothesis that specific bacterial strains associated with truffles can act as mycorrhizal helper bacteria, highlighting the potential for co-inoculation strategies to enhance quality of truffle-inoculated seedlings in nurseries. However, further research is needed to gain a deeper understanding of the interactions within the mycorrhizosphere that could contribute to improving nursery seedling quality.

BioTech doi: 10.3390/biotech14030068

Authors: Márta Balázs Izabella Péter Hunor Bartos Zsolt Bodor Emőke Antal Csilla Albert Ildikó Miklóssy

A continuous effort is needed to develop sustainable production methods for industrial platform chemicals. B. succiniciproducens, a natural succinic acid-producer, can metabolize five and six carbon atoms containing sugars in pure form as well as from agro-industrial wastes. In our work, we investigate the conversion of industrial by-products, apple pomace from apple juice production, and whey waste from milk processing to succinic acid and other organic acids (lactic, formic, and acetic acid). We obtained a succinic acid yield of 0.224 g/g total consumed fermentable sugars, lactic acid yield was 0.087 g/g, in turn, formic acid was produced at a 0.034 g/g yield, and acetic acid was obtained at 0.010 g/g total consumed fermentable sugars, using a thermal pretreated apple pomace-based medium. In the case of pretreated whey-based medium formulation, we obtained a succinic acid yield of 0.236 g/g consumed lactose, while formic acid and acetic acid were produced as well (0.09 g/g and 0.101 g/g, respectively). We demonstrate that lactose is a promising carbon source for organic acid production by B. succiniciproducens, while our study is the first to propose the use of a similarly available agro-industrial by-product, apple pomace, for the fermentative production of succinic acid by B. succiniciproducens.

BioTech doi: 10.3390/biotech14030067

Authors: Jakob Young Maliea Nipko Spencer Butterfield Zachary Aanderud

Extremophilic biological process (EBP) pretreatment increases substrate availability in anaerobic digestion, but the effect on downstream microbial community composition in industrial systems is not characterized. Changes in microbial communities were determined at an industrial facility processing dairy manure in a modified split-stream system with three reactor types: (1) EBP tanks at 70–72 °C; (2) mesophilic Continuously Stirred Tank Reactors (CSTRs); (3) mesophilic Induced Bed Reactors (IBRs) receiving combined CSTR and EBP effluent. All reactors had a two-day hydraulic retention time. Samples were collected weekly for 60 days. pH, volatile fatty acid and bicarbonate concentrations, COD, and methane yield were measured to assess tank environmental conditions. Microbial community compositions were obtained via 16S rRNA gene sequencing. EBP pretreatment increased acetate availability but led to a decline in the relative abundance of acetoclastic Methanosarcina species in downstream IBRs. Rather, syntrophic methanogens, e.g., members of Methanobacteriaceae, increased in relative abundance and became central to microbial co-occurrence networks, particularly in association with hydrogen-producing bacteria. Network analysis also demonstrated that these syntrophic relationships were tightly coordinated in pretreated digestate but absent in the untreated CSTRs. By promoting syntrophic methanogenesis while increasing acetate concentrations, EBP pretreatment requires system configurations that enable acetoclast retention to prevent acetate underutilization and maximize methane yields.

BioTech doi: 10.3390/biotech14030066

Authors: Sawichaya Orpool Suthaphat Kamthai Thanyaporn Siriwoharn Patompong Khaw-on Aree Deenu Srisuwan Naruenartwongsakul

Hemp (Cannabis sativa L.) seed is progressively emerging as an innovative and sustainable source of plant oil. Defatted hempseed meal is rich in protein and carbohydrates, which bacteria can convert into cellulose using glucose and fructose. The optimal conditions for bacterial cellulose (BC) production from hempseed meal were evaluated by investigating total solid concentrations ranging from 8 to 16 °Brix using Komagataeibacter nataicola under controlled conditions. The changes in pH, bioactive compounds, organic acids, and carbon source concentrations were monitored during the fermentation process. The highest yield of BC, 12.41 g/L, was obtained at 10 °Brix after 14 days of fermentation. It was found that the production of BC was negatively impacted by a decrease in pH and an increase in organic acids. BC exhibited a ribbon-like 3D network structure and a crystallinity index of about 70%, with excellent water-holding capacity, low oil-holding capacity, high emulsifying activity, and high emulsion stability (11.21%, 2.71%, 34.33%, and 39.11%, respectively). This BC possesses exceptional mechanical properties, a high degree of crystallinity, and superior water-holding capacity, making it valuable in various industries such as food, pharmaceuticals, and biotechnology.

BioTech doi: 10.3390/biotech14030065

Authors: Jagoš Golubović Damjan Vučurović

Neurosurgery is undergoing a significant transformation driven by advances in biomaterials and tissue engineering. These interdisciplinary innovations address challenges in repairing and regenerating neural tissues, integrating cranial and spinal implants, and improving patient outcomes. The incidence of neurological injuries such as traumatic brain injury and spinal cord injury remains high, underscoring the need for improved therapeutic strategies. This review provides a comprehensive overview of current biomaterial and tissue engineering approaches in neurosurgery, highlighting developments in neural tissue repair, cranial and spinal implants, spinal cord injury treatment, and peripheral nerve regeneration. Key challenges—such as ensuring biocompatibility, modulating the immune response, and bridging the gap between laboratory research and clinical application—are discussed. Emerging technologies including 3D bioprinting, nanotechnology (removing microfluidics), and microfluidics are examined for their potential to revolutionize neurosurgical treatments. The need for interdisciplinary collaboration among neurosurgeons, material scientists, and biologists is emphasized as critical for overcoming translational barriers and accelerating the clinical translation of these promising technologies.

BioTech doi: 10.3390/biotech14030064

Authors: Arisa Ando Hitomi Ohkubo Hisae Maki Takumi Nishiuchi Takumi Ogawa Tomofumi Mochizuki Daisaku Ohta Hiroaki Kodama Taira Miyahara

Transgrafting constitutes a technique involving the integration of genetically modified (GM) and non-GM plant organisms. Typically, edible components derived from non-GM scions are categorized as non-GM food products, attributed to the absence of exogenous genetic material within their respective genomes. Non-GM food status could be compromised if proteins translocated across the graft interface. We investigated the movement of insecticidal Bacillus thuringiensis (Bt) crystal proteins, widely utilized in GM crop species. Tobacco plants engineered to express the Cry1Ab gene exhibited trace levels of Cry1Ab protein accumulation. In transgrafted plants, translocated Cry1Ab protein originating from GM rootstocks was detectable within scion foliar tissues but not within the seeds obtained from the non-GM scion. This result unequivocally demonstrates the capacity for Bt protein translocation from rootstocks to scions yet indicates a constrained distribution confined to scion tissues relatively close to the graft junction. While regulatory considerations necessitate a thorough appraisal of potential risks associated with Bt proteins, the results shown here facilitate the commercialization of the edible components as non-GM food products.

BioTech doi: 10.3390/biotech14030063

Authors: El Hadj Hussein Tapily Kan Modeste Kouassi Marius Konan Kouassi John Steven S. Seka Fidèle Tiendrébéogo Justin S. Pita

Regenerating sweet potato from field-derived plant material requires careful management of several critical factors, including the effectiveness of the disinfectant, the age of the explant, and the genotype used. In this context, establishing a reliable aseptic protocol is essential for successful in vitro culture. This study aimed to assess the effects of two disinfectants (sodium hypochlorite and mercuric chloride), three sweet potato genotypes (Nakabo, Boyapleu, and Irene), and three explant ages (2, 3, and 4 weeks) on clean culture establishment and regeneration efficiency from nodal explants. The findings revealed that regeneration success is significantly influenced by the type and concentration of disinfectant, explant age, and genotype. Treatment with 10% sodium hypochlorite markedly reduced contamination, achieving clean culture and regeneration rates of 75.72 ± 3.36% and 86.83 ± 3.02%, respectively, regardless of explant age. In contrast, higher concentrations of mercuric chloride induced necrosis in the explants. The highest clean culture rate (93.82 ± 1.16%) was observed in 3-week-old explants, which also showed a regeneration rate of 54.93 ± 3.19%. Furthermore, the Boyapleu and Irene genotypes demonstrated good suitability for in vitro culture, whereas the Nakabo genotype performed poorly under the tested conditions.

BioTech doi: 10.3390/biotech14030062

Authors: Jéssica de Araujo Zanoni Izabela Karolina Costa Zilli Guilherme de Paula Pretto Flavio Augusto Vicente Seixas Marcela Marques de Freitas Lima Eliana Gertrudes de Macedo Lemos Eleni Gomes Gabriel Zazeri Gustavo Orlando Bonilla-Rodriguez

Industrial applications of xylanases in high-temperature settings are limited by enzyme instability. This study evaluated glycerol and phenolic compounds as modulators of the catalytic and structural properties of a recombinant Myceliophthora heterothallica endoxylanase (rMhXyn) expressed in Komagataella phaffii. Glycerol (20% v/v) significantly improved thermostability (5-fold increase in half-life at 55 °C), decreased the activation energy for catalysis, and enhanced structural rigidity as evidenced by molecular dynamics simulations (reduced RMSD and Rg). In contrast, phenolic acids provided only short-term stabilization at moderate temperatures and did not confer structural benefits. Enzyme kinetics revealed that glycerol enhanced catalytic turnover (↑Vmax), while phenolic compounds modified both K′ and cooperativity (Hill coefficient). Thermodynamic analysis supported glycerol’s stabilizing effect, with increased ∆H(D) and a positive shift in ∆S(D). These results suggest glycerol as a superior stabilizer for rMhXyn in high-temperature bioprocesses such as lignocellulosic biomass hydrolysis. These findings highlight the potential of targeted additives to improve enzyme performance for biotechnological applications.

BioTech doi: 10.3390/biotech14030061

Authors: Tuana Mendonça Faria Cintra Raquel Teles de Menezes Lara Steffany de Carvalho Leticia de Miguel Nazario Leandro Wang Hantao Maria Cristina Marcucci Luciane Dias de Oliveira Vanessa Marques Meccatti-Domiciano

Herbal medicines can be promising for the treatment of infections caused by multidrug-resistant microorganisms. This study aimed to evaluate Rosmarinus officinalis (Rosemary) hydroalcoholic extract (RHE) regarding its phytochemical composition and potential for eliminating polymicrobial biofilm of Candida albicans with multidrug-resistant bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa). The extraction and quantification of the extract (flavonoids and phenols) were performed, and its antioxidant activity (DPPH) and the presence of bio-active compounds were investigated using high-performance liquid chromatography with Diode Array Detection (HPLC-DAD) and Gas Chromatography–Mass Spectrometry (GC-MS). The minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) were determined, and the extract’s action on polymicrobial biofilms was evaluated using the MTT assay. Data were analyzed using one-way ANOVA and Tukey’s tests, as well as Kruskal–Wallis and Dunn’s tests, with a significance level of 5%. RHE showed compatible amounts of flavonoids and phenols, with an EC50 of 19.53 µg/mL. Through HPLC-DAD and GC-MS, biomolecules such as rosmarinic acid and α-Pinene were identified. The extract exhibited microbicidal activity and antibiofilm action, with reduction percentages of up to 69.6% (p < 0.05), showing superior performance compared to 0.12% chlorhexidine against C. albicans + A. baumannii. In conclusion, RHE may be a promising therapeutic agent against multidrug-resistant pathogens.

BioTech doi: 10.3390/biotech14030060

Authors: Elvira E. Ziganshina Ayrat M. Ziganshin

The study of microalgae has led to significant progress in recent decades. The current microalgal biomass yield is unsatisfactory, except for certain species that are cultivated for the nutraceutical and pharmaceutical industries. In this study, the growth efficiency and biochemical composition of Tetradesmus obliquus at high levels of nutrients were characterized. Increasing the NH4+-N content in the medium to 164 mg L−1 allowed the algae to steadily accumulate biomass (6.14 ± 0.28 g L−1) with a moderate content of starch. Optimizing the levels of N, P, and S allowed the biomass productivity to increase from the average 0.45 to 0.88 g L−1 day−1. A further increase of NH4+-N to 410 mg L−1 and other nutrients’ concentration allowed the algae to accumulate biomass (7.50 ± 0.28 g L−1), enriched with protein and pigments. The algae cultivated with the high load of nutrients reached 100%, 84%, and 96% removal of N, P, and S, respectively. Adding the NaHCO3 to the photobioreactor for pH adjustment (instead of NaOH) did not significantly improve the growth parameters or affect the composition of the algal cells. In general, our study will improve the comprehensive understanding of culture-based approaches to study the perspective use of the alga T. obliquus.

BioTech doi: 10.3390/biotech14030059

Authors: Francisco Cadena-Cadena Joe Luis Arias-Moscoso Leandris Argentel-Martínez Jony R. Torres Velazquez Dulce Alondra Cuevas-Acuña Nydia Estrellita Buitimea Cantua Bartolo Concha-Frías

This study evaluated the effect of ultrasonic pulse-assisted extraction on the yield and antioxidant activity of essential oils from grapefruit (Citrus paradisi) by-products using hydrodistillation and Soxhlet solvent extraction (hexane, acetone, ethanol). Ultrasound was applied at 40% amplitude for 20 min before extraction. Results showed that ultrasound significantly increased extraction yield with hexane (from 2.6 ± 0.58% to 7.6 ± 1.5%) and acetone (from 8.6 ± 0.96% to 12 ± 1.4%), while ultrasound-assisted hydrodistillation nearly doubled the yield (from 0.7 ± 0.03% to 1.5 ± 0.49%). In contrast, ultrasound decreased yield with ethanol by 3%. Antioxidant activity measured by TEAC assay was highest in acetone extracts without ultrasound (13,366.5 ± 7.66 mmol TE/g) and ethanol extracts (12,606.8 ± 0.51 mmol TE/g). However, ultrasound combined with ethanol increased DPPH scavenging activity from 1073.5 ± 1.07 µg/mL to 4933.3 ± 0.71 µg/mL and maintained high flavonoid content (9.41 ± 0.15 mg/mL) and phenolics (5.33 ± 0.09 mg/mL). Ultrasound-assisted hydrodistillation also enhanced antioxidant capacity, with DPPH values rising from 51.82 ± 5.56 µg/mL to 2413.03 ± 3.17 µg/mL. These findings demonstrate that ultrasound effectively enhances essential oil extraction and antioxidant activity depending on the solvent used, underscoring the potential of this clean technology for valorizing citrus by-products.

BioTech doi: 10.3390/biotech14030058

Authors: William G. Ryan V. Smita Sahay John Vergis Corey Weistuch Jarek Meller Robert E. McCullumsmith

In bioinformatics, pathway analyses are used to interpret biological data by mapping measured molecules with known pathways to discover their functional processes and relationships. Pathway analysis has become an essential tool for interpreting large-scale omics data, translating complex gene sets into actionable experimental insights. However, issues inherent to pathway databases and misinterpretations of pathway relevance often result in “pathway fails,” where findings, though statistically significant, lack biological applicability. For example, the Tumor Necrosis Factor (TNF) pathway was originally annotated based on its association with observed tumor necrosis, while it is multifunctional across diverse physiological processes in the body. This review broadly evaluates pathway analysis interpretation, including embedding-based, semantic similarity-based, and network-based approaches to clarify their ideal use-case scenarios. Each method for interpretation is assessed for its strengths, such as high-quality visualizations and ease of use, as well as its limitations, including data redundancy and database compatibility challenges. Despite advancements in the field, the principle of “garbage in, garbage out” (GIGO) shows that input quality and method choice are critical for reliable and biologically meaningful results. Methodological standardization, scalability improvements, and integration with diverse data sources remain areas for further development. By providing critical guidance with contextual examples such as TNF, we aim to help researchers align their objectives with the appropriate method. Advancing pathway analysis interpretation will further enhance the utility of pathway analysis, ultimately propelling progress in systems biology and personalized medicine.

BioTech doi: 10.3390/biotech14030057

Authors: Haiyan Wang Yanxing Yin Liu Wang Yifan Wang Xiaotong Liu Lijuan Shi

Maternal and child health during pregnancy is an important issue in global public health, and the classification accuracy of fetal cardiotocography (CTG), as a key tool for monitoring fetal health during pregnancy, is directly related to the effectiveness of early diagnosis and intervention. Due to the serious category imbalance problem of CTG data, traditional models find it challenging to take into account a small number of categories of samples, increasing the risk of leakage and misdiagnosis. To solve this problem, this paper proposes a two-step innovation: firstly, we design a method of adaptive adjustment of misclassification loss function weights (MAAL), which dynamically identifies and increases the focus on misclassified samples based on misclassification rates. Secondly, a primary and secondary correction network model (MAC-NET) is constructed to carry out secondary correction for the misclassified samples of the primary model. Experimental results show that the method proposed in this paper achieves 99.39% accuracy on the UCI publicly available fetal health dataset, and also obtains excellent performance on other domain imbalance datasets. This demonstrates that the model is not only effective in alleviating the problem of category imbalance, but also has very high clinical utility.

BioTech doi: 10.3390/biotech14030056

Authors: Raphael P. Moreira Marcelo R. Vicari Henrique A. Mulim Theresa M. Casey Jacquelyn Boerman Xing Fu Hinayah R. Oliveira

While bulk RNA sequencing provides a comprehensive view of transcriptomes, it lacks cell type specificity. Single-cell RNA sequencing (scRNA-seq) overcomes this limitation by providing detailed insights at the individual cell level, though it involves higher costs. Deconvolution methods can estimate cell type proportions in bulk RNA-seq data, but their results may vary based on the scRNA-seq reference data and software used. This study investigates the estimation of muscle fiber type proportions through deconvolution analysis of Longissimus dorsi muscle bulk RNA-seq data from late-gestation Holstein Friesian multiparous cows. Four software tools (i.e., CIBERSORTx, Cellanneal, DeconvR-NNLS, and DeconvR-RLM) were compared using scRNA-seq reference data from Brahman and Wagyu cattle breeds, which included proportions of types I, IIa, and IIx myofibers. Kruskal–Wallis and Dunn’s tests revealed that the breed of reference data significantly influenced the proportions of type IIa and IIx muscle fibers across different deconvolution methods. To the best of our knowledge, this is the first study to show that the cattle breed used in reference scRNA-seq data can substantially impact deconvolution outcomes, highlighting a critical consideration for accurate cell type proportion estimation in livestock genomics. These findings suggest that future deconvolution studies should carefully consider breed compatibility between reference and target datasets.

BioTech doi: 10.3390/biotech14030055

Authors: Isaac D. Raplee Samiksha A. Borkar Li Yin Guglielmo M. Venturi Jerry Shen Kai-Fen Chang Upasana Nepal John W. Sleasman Maureen M. Goodenow

Gene expression analysis is crucial in understanding cellular processes, development, health, and disease. With RNA-seq outpacing microarray as the chosen platform for gene expression, is there space for array data in future profiling? This study involved 35 participants from the Adolescent Medicine Trials Network for HIV/AIDS Intervention protocol. RNA was isolated from whole blood samples and analyzed using both microarray and RNA-seq technologies. Data processing included quality control, normalization, and statistical analysis using non-parametric Mann–Whitney U tests. Differential expression analysis and pathway analysis were conducted to compare the outputs of the two platforms. The study found a high correlation in gene expression profiles between microarray and RNA-seq, with a median Pearson correlation coefficient of 0.76. RNA-seq identified 2395 differentially expressed genes (DEGs), while microarray identified 427 DEGs, with 223 DEGs shared between the two platforms. Pathway analysis revealed 205 perturbed pathways by RNA-seq and 47 by microarray, with 30 pathways shared. Both microarray and RNA-seq technologies provide highly concordant results when analyzed with consistent non-parametric statistical methods. The findings emphasize that both methods are reliable for gene expression analysis and can be used complementarily to enhance the robustness of biological insights.

BioTech doi: 10.3390/biotech14030054

Authors: Prasika Arulrajah Sophi Katharina Riessner Anna-Lena Heins Dirk Weuster-Botz

Large-scale bioprocesses often suffer from spatial heterogeneities, which impact microbial performance and often lead to phenotypic population heterogeneity. To better understand these effects at the single-cell level, this study applied, for the first time, automated real-time flow cytometry (ART-FCM) to monitor L-phenylalanine production with an Escherichia coli triple reporter strain in a fed-batch process with glycerol as the carbon source. The strain was cultivated in both a well-mixed stirred-tank bioreactor (STR) and a scale-down two-compartment bioreactor (TCB), consisting of an STR and a coiled flow inverter (CFI) in bypass, to simulate spatial heterogeneities. ART-FCM enabled autonomous, high-frequency sampling every 20 min, allowing for real-time tracking of fluorescence signals linked to growth (rrnB-mEmerald), oxygen availability (narGHIJ-CyOFP1), and product formation (aroFBL-mCardinal2). The STR exhibited uniform reporter expression and higher biomass accumulation, while the TCB showed delayed product formation and pronounced phenotypic diversification depending on the set mean residence time in the CFI. Single-cell fluorescence distributions revealed that the shorter mean residence time in the CFI resulted in pronounced subpopulation formation, whereas longer exposure attenuated heterogeneity, indicating transcriptional adaptation. This finding highlights a critical aspect of scale-down studies: increased exposure duration to perturbations can enhance population robustness. Overall, this study demonstrates the relevance of ART-FCM, in combination with a multi-reporter strain, as a pioneering tool for capturing dynamic cellular behavior and correlating it to process performance, providing deeper insights into microbial heterogeneity under fluctuating bioprocess conditions.

BioTech doi: 10.3390/biotech14030053

Authors: Petya Marinova Denica Blazheva Aleksandar Slavchev Petia Genova-Kalou

This study investigates the cytotoxic properties of metal complexes incorporating thio-uracil derivatives, specifically 2,4-dithiouracil and 6-propyl-2-thiouracil. The research focuses on the cytotoxic effects of Cu(II) and Pd(II) complexes with 6-propyl-2-thiouracil, as well as mixed-ligand transition metal Cu(II) and Au(III) complexes of 2,4-dithiouracil with 2-thiouracil and uracil. Cytotoxic activity was assessed against human cervical carcinoma cells (HeLa) and normal kidney cells from the African green monkey. The results demonstrated that incorporating Cu(II) and Au(III) into the compound structures significantly enhanced their cytotoxic effects. Notably, all tested complexes exhibited a stronger inhibitory effect on cancer cell proliferation compared to normal cells, with the palladium(II) complex of 6-propyl-2-thiouracil showing the lowest CD50 value against the tumor cell line (0.00064 mM), which were 149 times lower than that of the ligand (0.0955 mM). These findings suggest that thio-uracil-based metal complexes, particularly those containing palladium (II) and gold(III), hold significant potential for further development as anticancer agents.

BioTech doi: 10.3390/biotech14030052

Authors: Vehary Sakanyan

Science has made significant progress in detecting reactive oxygen species (ROS) in tobacco smoke, which is an important step for precision cancer therapy. An important advance is also the understanding that superoxide can be produced by electrophilic molecules. The dual action of hydrogen peroxide, directly or via electrophilic molecules, in the development of oxidative stress allows for the identification of target proteins that can potentially stop unwanted signals in cancer development. However, despite advances in proteomics, reliable inhibitors to stop ROS-associated cancer progression have not yet been proposed for the treatment of tobacco cigarette smokers. This is likely due to an imperfect understanding of the diversity of molecular mechanisms of anti-ROS action. Fluorescent protein detection in living cells, called in-gel, offers a direct route to a better understanding of the rapid interaction of ROS and electrophilic compounds with targeted proteins. It seemed that the traditional paradigm of pharmaceutical innovation “one drug, one disease” did not solve the problem of tobacco smoking causing cancer. However, among the various therapeutic treatments for tobacco smokers, the best way to combat cancer today is smoking cessation, which fits into the “one-cure” paradigm.

BioTech doi: 10.3390/biotech14030051

Authors: Chaozheng Lu Guangxin Xu Yin Tian Zhiwei Yi Xixiang Tang

Fibronectin (FN), a primary component of the extracellular matrix (ECM), features multiple structural domains closely linked to various cellular behaviors, including migration, spreading, adhesion, and proliferation. The FN3 domain, which contains the RGD sequence, is critical in tissue repair because it enables interaction with integrin receptors on the cell surface. However, the large molecular weight of wild-type FN presents challenges for its large-scale production through heterologous expression. Therefore, this study focused on cloning the FN3 functional domain of full-length FN for expression and validation. This study selected Bacillus subtilis as the expression host due to its prominent advantages, including efficient protein secretion, absence of endotoxins, and minimal codon bias. The recombinant vector pHT43-FN3 was successfully constructed through homologous recombination technology and transformed into Bacillus subtilis WB800N. The FN3 protein was successfully expressed after induction with IPTG. Following purification of the recombinant FN protein using a His-tag nickel column, SDS-PAGE analysis showed that the molecular weight of FN3 was approximately 27.3 kDa. Western blot analysis confirmed the correct expression of FN3, and the BCA protein assay kit determined a protein yield of 5.4 mg/L. CCK8 testing demonstrated the good biocompatibility of FN3. In vitro cell experiments showed that FN3 significantly promoted cell migration at a 20 μg/mL concentration and enhanced cell adhesion at 10 μg/mL. In summary, this study successfully utilized Bacillus subtilis to express the FN3 functional domain peptide from FN protein and has validated its ability to promote cell migration and adhesion. These findings not only provide a strategy for the expression of FN protein in B. subtilis, but also establish an experimental foundation for the potential application of FN3 protein in tissue repair fields such as cutaneous wound healing and cartilage regeneration.

BioTech doi: 10.3390/biotech14030050

Authors: Felipe Roberto Flores-de la Rosa Cristian Matilde-Hernández Nelly Abigail González-Oviedo Humberto José Estrella-Maldonado Liliana Eunice Saucedo-Picazo Ricardo Santillán-Mendoza

Vanilla is a highly valuable spice in multiple industries worldwide. However, it faces a serious problem due to a disease known as root and stem rot, caused by the fungus Fusarium oxysporum f. sp. vanillae. Little is known about the pathogenicity mechanisms this fungus employs to establish the disease, making it imperative to elucidate mechanisms such as the presence of pathogenicity effectors in its genome. The aim of the present study was to determine the presence of the SIX gene family in the genome of three strains of F. oxysporum associated with root rot: two pathogenic strains and one non-pathogenic endophyte strain. Additionally, the complete effectorome of these strains was predicted and compared to exclude effectors present in the endophytic strain. Our results show that only the SIX9 gene is present in the strains associated with the disease, regardless of their pathogenic nature. Furthermore, no variation was observed in the SIX9 gene among these strains, suggesting that SIX9 is not involved in pathogenicity. Instead, we identified 339 shared effectors among the three strains, including the non-pathogenic strain, strongly suggesting that these genes are not relevant for establishing root rot but may play a role in endophytic colonization. The highly virulent strain IXF41 exhibited eight exclusive pathogenicity effectors, while the moderately virulent strain IXF50 had four. Additionally, one effector was identified as shared between these two strains but absent in the endophytic strain. These effectors and their promoters were characterized, revealing the presence of several cis-regulatory elements responsive to plant hormones. Overall, our findings provide novel insights into the genomic determinants of virulence in F. oxysporum f. sp. vanillae, offering a foundation for future functional studies and the development of targeted disease management strategies.

BioTech doi: 10.3390/biotech14020049

Authors: Simona Neagu Mihaela Marilena Stancu

Microorganisms from saline environments have garnered significant interest due to their unique adaptations, which enable them to thrive under high-salt conditions and synthesize valuable biomolecules. This study investigates the biosynthesis of biomolecules, such as extracellular hydrolytic enzymes, biosurfactants, and carotenoid pigments, by four newly halotolerant bacterial strains isolated from saline environments in the Băicoi (soil, water) and Curmătura (mud) area (Prahova County, Romania). Isolation was performed on two selective culture media with different NaCl concentrations (1.7 M, 3.4 M). Based on their phenotypic and molecular characteristics, the four halotolerant bacteria were identified as Halomonas elongata SB8, Bacillus altitudinis CN6, Planococcus rifietoensis CN8, and Halomonas stenophila IB5. The two bacterial strains from the Halomonas genus exhibited growth in MH medium containing elevated NaCl concentrations (0–5 M), in contrast to the other two strains from Bacillus (0–2 M) and Planococcus (0–3 M). The growth of these bacteria under different salinity conditions, hydrocarbon tolerance, and biomolecule production were assessed through biochemical assays, spectrophotometry, and high-performance thin-layer chromatography. The antimicrobial properties of biosurfactants and carotenoids produced by H. elongata SB8, B. altitudinis CN6, P. rifietoensis CN8, and H. stenophila IB5 were evaluated against four reference pathogenic microorganisms from the genera Escherichia, Pseudomonas, Staphylococcus, and Candida. H. elongata SB8 showed the highest hydrocarbon tolerance. B. altitudinis CN6 exhibited multiple hydrolase activities and, along with H. elongata SB8, demonstrated biosurfactant production. P. rifietoensis CN8 produced the highest carotenoid concentration with antifungal and antimicrobial activity. Exploring these organisms opens new pathways for bioremediation, industrial bioprocessing, and sustainable biomolecule production.

BioTech doi: 10.3390/biotech14020048

Authors: Emily Walter Akshaya Biswal Peggy Ozias-Akins Ye Chu

Interspecific and intersectional crosses have introduced valuable genetic traits for blueberry (Vaccinium sect. Cyanococcus) cultivar improvement. Introgression from Vaccinium species at the diploid, tetraploid, and hexaploid levels has been found in cultivated blueberries. Continued efforts to integrate wild blueberry genetic resources into blueberry breeding are essential to broaden the genetic diversity of cultivated blueberries. However, performing heteroploid crosses among Vaccinium species is challenging. Polyploid induction through tissue culture has been useful in bridging ploidy barriers. Mixoploid or chimeric shoots often are produced, along with solid polyploid mutants. These chimeras are mostly discarded because of their genome instability and the difficulty in identifying periclinal mutants carrying germline mutations. Since induced polyploidy in blueberries often results in a low frequency of solid mutant lines, it is important to recover solid polyploids through chimera dissociation. In this study, two vegetative propagation methods, i.e., axillary and adventitious shoot induction, were evaluated for their efficiency in chimera dissociation. Significantly higher rates of chimera dissociation were found in adventitious shoot induction compared to axillary shoot induction. Approximately 89% and 82% of the adventitious shoots induced from mixoploid lines 145.11 and 169.40 were solid polyploids, respectively, whereas only 25% and 53% of solid polyploids were recovered through axillary shoot induction in these lines. Effective chimera dissociation provides useful and stable genetic materials to enhance blueberry breeding.

BioTech doi: 10.3390/biotech14020047

Authors: Hanyu Chu Lijie Zhou Yanzhen Mao Ren Liu Jiaojiao Han Xiurong Su Jun Zhou

Lacticaseibacillus rhamnosus (L. rhamnosus) is a safe probiotic with no side effects, providing benefits such as gut microbiota regulation and immune enhancement, making it highly valuable with strong potential. However, strains from different sources have unique traits, and whole-genome sequencing (WGS) helps analyse these differences. In this study, we used WGS to examine L. rhamnosus strains from mice with fish oil-treated smoking-induced pneumonia to better understand their biological functions and explore possible anti-inflammatory mechanisms. Methods: We isolated a strain, Lacticaseibacillus rhamnosus CP-1 (L. rhamnosus CP-1), from mice intestines where fish oil alleviated smoking-induced pneumonia. Identification of probiotic-related genes by WGS and characterised the strain’s probiotic properties. Results: L. rhamnosus CP-1 has a single circular chromosome (2,989,570 bp, 46.76% GC content) and no plasmids. COG, GO, and KEGG databases revealed genes linked to carbohydrate metabolism. The CAZy database identified GH25 lysozyme and PL8 polysaccharide lyase genes. KEGG highlighted an antimicrobial peptide ABC transporter permease, while TCDB noted the ABC-type antimicrobial peptide transporter (the main active transport component). KEGG also showed 10 genes for terpenoid skeleton biosynthesis and 5 for keto-glycan unit biosynthesis. Additionally, L. rhamnosus CP-1 carries metabolic regulators and bacteriocin-related genes. Conclusions: Whole-genome sequencing analysis revealed that L. rhamnosus CP-1 has carbohydrate utilisation and potential anti-inflammatory effects at the molecular level. Potential functional genes include carbohydrate transport and hydrolase, antimicrobial peptide ABC transporter and its osmotic enzyme components, bacteriocin immune protein, terpenoid skeleton, and keto-glycan synthesis.

BioTech doi: 10.3390/biotech14020046

Authors: Somruthai Kaeoboon Rattanaporn Songserm Rungcharn Suksungworn Sutsawat Duangsrisai Nuttha Sanevas

Microalgae represent promising biotechnological platforms for bioactive compound production with pharmaceutical applications. This study investigated the phytochemical composition and biological activities of lipid extracts from three Chlorella species to evaluate their potential as antioxidant and antidiabetic sources. Lipid extraction using chloroform–methanol (2:1) followed by GC–MS analysis revealed distinct compound distributions: 29 compounds in C. ellipsoidea, 33 in C. sorokiniana, and 19 in C. vulgaris. Major bioactive compounds included 2-hexanol, 1,3,6-heptatriene, 4-(2,3-dimethyl-2-cyclopenten-1-yl)-4-methylpentanal, n-hexadecanoic acid, and octadecanoic acid. Biological activity screening encompassed antioxidant assessment through DPPH• and •NO radical scavenging assays and FRAP analysis, while antidiabetic potential was evaluated using α-glucosidase and α-amylase inhibition assays. C. sorokiniana exhibited superior bioactivity with the highest antioxidant capacity (DPPH• IC50 = 329.03 ± 4.30 µg/mL; •NO IC50 = 435.53 ± 10.20 µg/mL; FRAP = 94.74 ± 5.72 mg TE/g) and strongest enzyme inhibition (α-glucosidase IC50 = 752.75 ± 57.95 µg/mL; α-amylase IC50 = 3458.50 ± 104.01 µg/mL). This is the first report on C. sorokiniana strain KU.B2′s biological properties and phytochemical profile. These findings establish C. sorokiniana as a valuable biotechnological platform for pharmaceutical bioactive compound development.

BioTech doi: 10.3390/biotech14020045

Authors: José Luis Aguirre-Noyola Marco A. Ramírez-Mosqueda Jorge David Cadena-Zamudio José Humberto Caamal-Velázquez Esmeralda J. Cruz-Gutiérrez Alma Armenta-Medina

Nanobiotechnology applications in plant tissue culture have improved the development and physiology of explants, resulting in plants with high genetic homogeneity and phytosanitary quality. Silver nanoparticles (AgNPs) are well-known for their microbicidal properties, but their biochemical effects on plants require further exploration. In this work, green-synthesized AgNPs were evaluated in strawberry in vitro culture, photosynthetic pigment production, and acclimatization. AgNPs produced by Lysinibacillus fusiformis were characterized. Strawberry explants were grown in vitro on MS medium with 0, 100, 200, and 300 mg L−1 AgNPs at 24 ± 2 °C and a photoperiod of 16:8 h light/dark. Shoot height and number, number of leaves, number of roots, and root length were evaluated, and chlorophyll (a, b, and total) was quantified. Rooted shoots were acclimatized ex vitro on substrates containing 0 and 200 mg L−1 AgNPs. The results showed that low AgNPs concentrations had a positive impact on shoot multiplication, development, and rooting, but at higher concentrations, the effects decayed. However, chlorophyll production improved with increasing AgNP concentration. Shoots treated with AgNPs showed higher ex vitro survival. Our study has direct implications for the profitability and sustainability of commercial strawberry production.