Cannabis sativa L. produces cannabinoids as high-value secondary metabolites within the specialized glandular trichomes of mature inflorescences. Large-scale cannabinoid production in conventional agriculture is limited due to extensive land requirements, irrigation requirements, pests and diseases, and reliance on agrochemicals. In vitro platforms offer a reliable and climate-independent alternative for the production of plant-derived compounds. However, cannabinoid biosynthesis in undifferentiated callus tissues remains poorly understood because these tissues lack specialized morphological structures. This study presents an innovative strategy to induce and sustainable production of cannabinoids in C. sativa callus cultures using biogenic silver nanoparticles (AgNPs) synthesized via Yarrowia lipolytica. After treatment of C. sativa L. callus cultures with biogenic AgNPs, cannabinoids (THC and CBD) were quantified by HPLC and the expression of key biosynthetic genes (CBDAS and THCAS) was analyzed by qPCR. In the control samples, cannabinoids were not detectable. Treatment with 20 mg/L AgNPs led to accumulation of THC (0.31
Spider silks are attractive materials due to their impressive material properties and their ability to self-assemble into a variety of morphological biomaterials for use as carriers for drug delivery. Previously, the M4R2 silk particles, self-assembled from recombinant spider silk protein, have demonstrated efficient loading capacity for positively charged antitumor polypeptides. However, the colloidal stability of these silk particles and their affinity for drugs still require improvement. In this study, we designed and produced a new bioengineered spider silk protein (DM4R2) by incorporating an aspartic acid residue. The DM4R2 particles exhibited a more negative charge and greater stability compared to M4R2 particles. Meanwhile, the DM4R2 particles showed no cytotoxicity and possessed a similar secondary structure composition to that of M4R2 particles. Although MTX and lysozyme were efficiently loaded onto both types of particles with up to 90
Polyhydroxybutyrate (PHB) is a biodegradable alternative to petrochemical plastics; however, its high production cost, largely attributed to the use of conventional carbon sources such as glucose, remains a major barrier. This study was designed to screen PHB-producing bacteria from dumping soil and evaluate the potential of sugarcane molasses, an agro-industrial waste, as a low-cost carbon source for PHB production. The PHB-positive isolate GSBB-5a2-2 was screened by using Sudan Black B and Nile Blue A staining techniques. The strain was identified as Pseudomonas plecoglossicida MK-6 based on partial 16 S rRNA gene sequencing (Gen Bank accession number. OQ236410). The optimal fermentation conditions were determined using a one-variable-at-a-time optimization approach. The maximum PHB production (7.3 g/L) was achieved with sugarcane molasses as the carbon source. A concentration of 5
In this study, we applied a semi-rational engineering strategy to improve the catalytic performance and operational stability of germacrene A synthase from Cichorium intybus (CiGASlo), a key biocatalyst for stereochemically defined (–)-β-elemene biosynthesis. An AlphaFold2-predicted model was first validated by confidence-score analysis, stereochemical-quality assessment, and structural comparison with a related plant germacrene A synthase. Molecular docking was then used to define substrate-proximal residues around farnesyl pyrophosphate (FPP), while alanine scanning and HotSpot Wizard 3.0 were used as stability- and mutational-tolerance filters rather than as direct predictors of catalytic activity. Experimental screening of a focused mutation library identified several variants with improved activity, including D337H, L415W, D477H, E485Y, R488A, W305A, and the double variant W305A/L415W. Experimental screening identified W305A/L415W as the variant with the highest specific activity under the fixed assay conditions (0.95 ± 0.03 U·mg− 1; 69.6
Leukoreduction filters (LRFs) are routinely used in blood transfusion services and retain substantial numbers of nucleated cells during blood processing. These discarded filters have therefore been explored as a potential source of peripheral blood-derived cells for endothelial progenitor cell (EPC)-like related in vitro studies. Leukoreduction filters were collected from the Iranian Blood Transfusion Organization (IBTO). Entrapped cells were recovered using a washing-based procedure without the use of magnetic-activated cell sorting (MACS) or endothelial differentiation media. A coating solution containing plasma-derived components, including cryoprecipitate and platelet lysate, was applied to improve early cell adhesion during in vitro culture. Cultured cells were characterized by flow cytometry using CD133 as a surface-associated progenitor marker. Approximately 70
The correctness and efficiency of the adapter connection are important factors affecting the quality and yield of the libraries in next-generation sequencing (NGS). Lengthening the sticky ends of the adapters and the targets can effectively improve the efficiency of ligation, however, it may cause a new trouble of adapter self-ligation. In this study, a strategy based on nickase is designed to limit or abolish long sticky-end adapters self-ligation. We firstly explored the enzymatic characteristics of Nt.BsmAI, a typical nickase. Then based on the working properties of Nt.BsmAI to design Y-adapters for NGS and evaluated the efficiency of this strategy for reducing long sticky-end adapter dimers. Moreover, the feasibility of Nb.BtsI (another nickase) was verified using the same strategy. This study provides new application for nickase in NGS library construction, and the possibility of using multi-base sticky end adapters in the future. NGS adapters with long sticky-ends paves a new avenue for enhancing the application performance of NGS, particularly for methylation sequencing, in biomedicine, with a higher efficiency in library preparation.
Prodigiosin is a natural red pigment produced by Serratia marcescens that has attracted attention due to its various biological activities and potential for industrial applications. In recent years, research aimed at producing this compound using highly efficient and economically viable bioprocesses has gained increasing importance. In this study, the OD600 value and prodigiosin production of Serratia marcescens isolated from onion were experimentally tested under varying environmental conditions (glycerol, pH, temperature, and time), and artificial neural network (ANN) and ANFIS models were designed to predict prodigiosin production using the obtained data. First, the red pigment extracted with acidified methanol was characterized using a UV-vis spectrophotometer and exhibited maximum absorbance at 535 nm. Next, the modeling phase began, and both the ANN and ANFIS architectures were trained using 80
Fungal inulinase hydrolyzes inulin to fructose, a commercially important intermediate with a wide range of applications in various industries. However, its industrial utilization is limited due to high cost of production and processing. Therefore, the present study reports the valorization of low-cost agricultural residues (AR) as an alternative substrate for inulinase production and its application in generation of fructose. In the study, an inulinase producing fungal strain was identified as Talaromyces domesticus ITCC 9483 based on preliminary and secondary screening. Further, various low-cost AR were screened for efficient inulinase production. Amongst these substrates, rice husk (RH) supported the highest enzymatic activity and was utilized as a major component for medium optimization in the presence of inulin as an inducer. It resulted in 2.02-fold increase in inulinase activity from 8.34 ± 1.45 U/mL to 16.86 ± 1.29 U/mL. The inulinase was purified resulting in 5.52-fold purification and a molecular mass of 45–50 kDa. The zymographic analysis confirmed inulinase via distinct red band on native gel. The purified inulinase exhibited maximum stability at pH 5.0 and 50 °C after 24-hours of incubation, with Km 0.83 ± 0.19 mM and Vmax 4.58 ± 0.48 µmol/min. The structural characteristics were evaluated using UV spectrum, FTIR and XRD. The optimized inulinase was further employed for valorization of AR generating maximum reducing sugar yield of 2.14, 0.96 and 0.34 mg/mL from WB, SR and wheat straw (WS). The high-performance liquid chromatography (HPLC) confirmed fructose as primary hydrolysis product with concentrations of 47.94, 38.91 and 28.99
Epsilon-poly-L-lysine (ε-PL), an antimicrobial peptide synthesized by Streptomyces albulus through submerged fermentation, is biosynthesized via multiple metabolic pathways. This suggests that global metabolic regulation and interspecies interactions contribute significantly to enhancing ε-PL yield. Current genetic and metabolic engineering strategies, however, primarily aim at strengthening specific metabolic fluxes, without adequately coordinating the diverse physiological requirements involved in ε-PL production. Previous research has shown that interspecies stress induced by Streptomyces gilvosporeus can elicit a broad enhancement of ε-PL biosynthetic pathways. Building on this finding, the present study seeks to identify key transcriptional regulators that respond to such interspecies stress and are capable of systematically improving metabolic efficiency. Comparative transcriptomic analysis revealed that transcription factors from the RegX3, LysR, and TetR families were significantly upregulated in S. albulus under interspecies stress. Corresponding overexpression strains (OE-RegX3, OE-LysR, OE-TetR1, and OE-TetR2) were constructed using a one-step cloning strategy, all exhibiting altered colony morphology. Among them, OE-LysR showed a 60.9
Soybeans are an important agricultural product, but they contain a significant amount of raffinose, which can cause flatulence when fermented by intestinal microorganisms. Invertase InvDz13 from Microbacterium trichothecenolyticum can effectively hydrolyze raffinose in soybean into the prebiotic melibiose, improving the nutritional value and economic benefits of soybean derivatives. However, its low yield limits industrial applications. This study produced InvDz13 in Bacillus subtilis, resulting in a recombinant enzyme with the natural enzyme’s excellent properties. The extracellular InvDz13 production in B. subtilis was enhanced by boosting synthesis and secretion in a suitable chassis cell. Various extracellular protease-deficient chassis cells were constructed, and it was found that Vpr and WprA enhance the extracellular InvDz13 activity. The recombinant InvDz13 production was further enhanced by sequentially engineering the promoter, screening the signal peptide, and regulating key elements in the secretion pathway. The recombinant strain WBSS142I4PA, constructed using the promoter PspoVG-PspoVG142 and signal peptide SPYomL, co-expressing signal peptide peptidases SppA, exhibited a 10.02-fold increase in extracellular InvDz13 activity in shake flasks. Notably, the extracellular InvDz13 activity of WBSS142I4PA in a 3-L fermenter reached 568.86 U/mL. To our knowledge, this is the highest recombinant expression level of an exogenous invertase in B. subtilis reported so far. This is the first study to express InvDz13 in B. subtilis and develop a novel multidimensional combinatorial strategy to enhance its extracellular production. This study provides a theoretical foundation for the large-scale application of InvDz13 in soybean processing and paves novel avenues for protein production in B. subtilis.
In silico approaches are becoming increasingly important for enhancing the affinity of antibodies and antibody fragments, including single-chain variable fragments (scFvs). In this study, we present an in silico affinity-maturation workflow designed to improve the affinity of an scFv directed against Neisseria meningitidis factor H-binding protein (fHbp) by integrating in silico maturation, molecular dynamics (MD) simulations, and experimental validation. Approximately 300 single-point substitutions were designed at scFv residues predicted to interact with fHbp, and the resulting V3 (R99D) scFv–fHbp complexes were analyzed through all-atom MD simulations. Guided by bioinformatic analyses, the top V3 (R99D) variant was cloned and expressed, and its binding activity was validated by ELISA. MD analyses identified five variants, Y35H, L31R, R99D, R99H, and A100K, that exhibited markedly enhanced complex stability. In all variants, the scFv secondary structure remained intact, whereas fHbp showed shrinkage relative to the scFv in the V3 (R99D)–fHbp complexes during the simulations. Hydrogen-bonding patterns and inter-residue distances further supported the improved affinity of the variants relative to the native form. The R99D variant showed the highest binding affinity, with an experimentally measured value of 67.93 × 10⁹ M⁻¹. Collectively, these findings underscore affinity maturation as a powerful strategy for engineering high-affinity antibody fragments.
Knee trauma is an important risk factor for post-traumatic osteoarthritis (PTOA), a progressive joint disease characterized by articular cartilage degeneration and limited disease-modifying treatment options. Matrix metalloproteinase-13 (MMP-13/collagenase-3) contributes to cartilage extracellular matrix degradation through cleavage of type II collagen, making it a relevant molecular target for cartilage-protective drug discovery. This study aimed to identify and prioritize potential small-molecule MMP-13 inhibitors using an integrated in silico workflow. The workflow included MMP-13 sequence retrieval, structural characterization, pharmacophore-based screening, molecular docking, ADMET/toxicity prediction, protein-ligand interaction analysis, protein-protein interaction and co-expression network analysis, and 100 ns molecular dynamics simulation. Among the screened compounds, P4C-BM was prioritized as an early-stage computational lead candidate based on its combined docking score, predicted ADMET profile, interaction pattern, and molecular dynamics stability. However, the findings are preliminary and computational only. The present study does not experimentally demonstrate direct MMP-13 inhibition, selectivity against other MMP family members, cartilage repair, clinical efficacy, or synergy with platelet-rich plasma, physiotherapy, or other treatment modalities. Among the screened compounds, P4C-BM was prioritized as an early-stage computational lead candidate based on its combined docking score, predicted ADMET profile, interaction pattern, and molecular dynamics stability. However, the findings are preliminary and computational only. The present study does not experimentally demonstrate direct MMP-13 inhibition, selectivity against other MMP family members, cartilage protection, cartilage repair, disease modification, or clinical efficacy. Therefore, P4C-BM should be regarded only as a candidate for further experimental validation in enzymatic assays, chondrocyte models, cartilage explant studies, and in vivo osteoarthritis or post-traumatic osteoarthritis models. No practice-related or treatment recommendations can be made from the present data.
This research provides the first extensive breed‑pool whole‑genome sequencing (WGS) analysis across five Egyptian sheep populations: Barki (BAR), Rahmani (RAH), their crossbred offspring (CRS), Ossimi (OSI) and Awassi (AWI). To establish a genomic atlas of the genetic architecture of production and adaptation in Egyptian sheep, providing a baseline for future candidate gene discovery and conservation strategies. Through Illumina sequencing of 120 samples, we compiled a dataset exceeding 470 Gb, with mean coverage depths spanning 24.2x to 41.3x. Variant profiling, functional annotation, KEGG pathway analysis, and independent structural variant analysis were conducted. Phenotypic data were collected and validated through qRT-PCR gene expression analysis. Variant profiling revealed between 11.9 and 17.4 million SNPs per breed after stringent filtering. Heterozygosity patterns (population‑level estimates) differed substantially between groups, recorded at 60.41
This study developed a dual-functional 3D-printed poly(ε-caprolactone) (PCL) scaffold loaded with quercetin (Q-PCL) for synergistic inhibition of bone metastatic tumors and promotion of bone regeneration. The scaffold was fabricated via melt extrusion, exhibiting a uniform porous structure conducive to sustained quercetin release. In vitro, Q-PCL significantly suppressed renal carcinoma (RENCA) cell proliferation and induced apoptosis, while enhancing the osteogenic differentiation and mineralization of bone marrow mesenchymal stem cells (BMSCs). In a murine subcutaneous tumor model, Q-PCL implantation effectively inhibited tumor growth via apoptosis induction without systemic toxicity. In a rat femoral defect model, the scaffold markedly accelerated bone repair, showing increased bone volume, improved trabecular morphology, and mature bone formation. The Q-PCL scaffold demonstrates great potential as a localized co-therapy strategy for treating osteolytic bone metastases and facilitating bone regeneration.
This study investigated the antioxidant and anticancer activities of selenium nanoparticles stabilized by exopolysaccharides from Pseudoduganella armeniaca ZMN-3 (EPS‑SeNPs). The synthesized EPS‑SeNPs had an average size of 145 nm and were thoroughly characterized. In vitro antioxidant assays revealed that at 4 mg/mL, EPS‑SeNPs scavenged 86.49 ± 0.53
Transgenic crops undergo rigorous safety assessments prior to commercialization, with molecular characterization serving as a critical component of regulatory review. This process establishes the identity, copy number, sequence integrity, absence of unintended foreign DNA, and insert stability across breeding generations. While whole-genome sequencing (WGS) has emerged as a powerful alternative to Southern blotting, the lack of accessible interpretation frameworks can be an entry barrier to those who wish to understand this modernized experimental setup. We developed an analytical workflow based on mapped-read signatures to characterize T-DNA (transfer DNA) insertions using short-read WGS data. Simulated Illumina paired-end datasets representing diverse transformation outcomes were generated and analyzed to define five informative read classes, which when observed mapped to a reference transformation construct provide distinct signatures indicating transformation outcomes. These signatures were applied to identify insertion boundaries, copy number, structural anomalies, and potential contamination. Mapped-read signatures can reliably distinguish single-copy inserts, multiple insertions, backbone co-integrations, and structural rearrangements, aided by coverage profiles and mate-pair orientations. We present representative examples and a practical interpretation to guide practitioners new to WGS-based molecular characterization and regulators assessing these data. This framework standardizes interpretation of short-read paired-end WGS data for molecular characterization without prescribing specific software. The platform-agnostic approach ensures broad applicability while enhancing transparency in regulatory assessments.
The genetic classification of microbial populations at high taxonomic resolution is crucial for clinical diagnosis and treatment. The classification of complex bacterial communities in the human gut was examined in this study by utilizing amplicon sequencing of the 16S ribosomal RNA (rRNA) gene. The influence of different sequencing platforms and amplicon regions on this classification was investigated. Nineteen human fecal samples were analyzed using both Nanopore MinION and Illumina MiSeq platforms.The main objective of the analysis was to assess how effectively these platforms can characterize gut microbiota at the Amplicon Sequence Variant (ASV), genus, and species levels, taking into account various amplicon regions within the 16S rRNA gene. The findings reveal significant disparities between the two platforms. In particular, the MinION platform demonstrates higher values in terms of ASV and species richness, as well as diversity (both alpha and beta diversity) compared to the MiSeq platform. These variations are dependent on the specific targeted amplicon region of the 16S rRNA gene. However, despite these differences in richness and diversity, there is a notable level of consistency observed in detecting the presence of dominant microbial taxa across both platforms, although their relative abundances varied significantly. Notably, the study emphasizes that the choice of primers used for amplification exerts the most significant impact on the classification results at the genus level, surpassing the influence of the sequencing platform itself. This research provides valuable insights into the strengths and limitations of long-read and short-read sequencing in the context of classifying complex gut microbiota, particularly in clinical microbiology. • MinION excels in novel sequence detection with increasing sequencing depth. • Both MinION and MiSeq identify high-abundance genera in the microbial community. • Primers’ impact overshadows sequencing platforms’ in V1-V9 region.
Fusobacterium nucleatum (F. nucleatum) is an oral commensal bacterium that acts as a pathobiont with pro-tumorigenic activity in various gastrointestinal cancers. However, its functional role, invasive capacity, and mechanistic contributions in cervical cancer remain largely unexplored. We identified F. nucleatum in cervical cancer tissues using bioinformatics and clinical 16S rRNA sequencing. Its spatial localization and intracellular presence were confirmed by fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM), respectively. Functional validation included in vitro assays for proliferation, migration, and apoptosis in cervical cancer cell lines, with bacterial invasion visualized by confocal microscopy, and in vivo tumor growth assessment in a xenograft model. The underlying mechanism involving high mobility group box 1 (HMGB1) and the NF-κB pathway was analyzed by western blot, qPCR, immunofluorescence, and ELISA. F. nucleatum was enriched in cervical cancer and correlated with poor patient survival. It invaded cervical cancer cells, promoted proliferation, migration, and invasion, suppressed apoptosis in vitro, and accelerated tumor growth in vivo. Mechanistically, infection triggered HMGB1 upregulation and specific activation of the canonical NF-κB pathway (via IκBα degradation, p65 phosphorylation/nuclear translocation), leading to selective secretion of IL-6/IL-8. Our study suggests that F. nucleatum is associated with cervical cancer malignancy, potentially acting through upregulation of HMGB1 and activation of the canonical NF-κB signaling pathway, thereby contributing to an altered tumor microenvironment. These findings reveal a previously unrecognized microbial-driven oncogenic mechanism in cervical cancer and highlight its potential as a prognostic marker and a therapeutic target. Not applicable in our manuscript.
