Search Results (479)

Candida albicans is a major fungal pathogen associated with vulvovaginal candidiasis, and rapid, sensitive detection remains challenging, particularly in amplification-free formats. Here, we report NaPddCas, a microfluidic-free, droplet-based CRISPR/Cas12a detection strategy for qualitative identification of Candida albicans DNA. Unlike conventional bulk CRISPR assays, NaPddCas partitions the reaction mixture into vortex-generated polydisperse droplets, enabling spatial confinement of Cas12a activation events and effective suppression of background fluorescence. This compartmentalization substantially enhances detection sensitivity without nucleic acid amplification or microfluidic devices. Using plasmid and genomic DNA templates, NaPddCas achieved reliable detection at concentrations several orders of magnitude lower than bulk CRISPR/Cas12a reactions. The assay further demonstrated high specificity against non-target bacterial and fungal species and was successfully applied to clinical vaginal secretion samples. Importantly, NaPddCas is designed as a qualitative or semi-qualitative droplet-dependent digital detection method rather than a quantitative digital assay. Owing to its simplicity, sensitivity, and amplification-free workflow, NaPddCas represents a practical approach for laboratory-based screening of Candida albicans infections. Full article

Lipid nanoparticles (LNPs) are now the go-to method for delivering genetic medicines, backed by real-world use in patients. Things like which fats they are made of, their shape at the molecular level, how ingredients mix, and how they are built, matter a lot. This review attempts to take a close look at how different components, such as ionizable lipids, auxiliary lipids (DSPC, DOPE), cholesterol, and PEG-based lipids, affect the bioavailability of LNPs. It also focuses on key functions of LNPs, including packaging genetic material, escaping cellular traps, spreading in the body, and remaining active in the blood. New data show that lipids with the right handedness and highly sensitive chiroptical quality control can sharpen delivery accuracy and boost transport rates, turning stereochemistry into a practical design knob. Rather than simply listing results, we examine real-world examples that are already used to regulate gene expression, enhance mRNA expression, splenic targeting, and show great potential for gene repair, protein replacement, and DNA base-editing applications. Also, recent advances in AI-based designs for LNPs that take molecular shape into account and help speed up modifications to lipid arrangements and mixture configurations are highlighted. In summary, this paper presents a practical and scientific blueprint to support smarter production of advanced LNPs used in genetic medicine, addressing existing obstacles, balanced with future opportunities. Full article

The growing demand for robust food authentication methods has driven the establishment of fast, sensitive, and field-based detection systems for identifying meat species. This study presents a colorimetric-based PSR approach for identifying yak (Bos grunniens) meat within fresh, thermally processed, and blended meat samples. Targeting the mitochondrial D-loop locus, the assay incorporates a simple alkaline lysis (AL) procedure for efficient DNA extraction, eliminating the requirement for specialized instrumentation. The PSR assay demonstrated high specificity, showing no evidence of cross-reactivity with closely associated food animals such as buffalo, cattle, goat, sheep, mithun, and pig. Sensitivity assessment revealed the assay’s capability to detect 1 pg of yak DNA, with reliable performance in samples exposed to thermal conditions up to 121 °C. Additionally, the technique detected yak meat down to a concentration of 0.1% in binary beef mixtures. This method provides a significant improvement in sensitivity over end-point PCR and is particularly well-suited for field applications due to its practical simplicity, affordability, as well as no reliance on sophisticated instrument. This is, to the best of our understanding, the first reported PSR-based approach developed for the identification of yak meat, offering a robust tool for food origin verification, regulatory enforcement, and product integrity monitoring. Full article

Lupin (Lupinus spp.) is increasingly incorporated into processed foods as a gluten-free ingredient and alternative protein source, but it is also a regulated allergen in the European Union due to cross-reactivity with other legumes, especially peanut. Reliable methods for detecting undeclared lupin traces in complex food matrices are therefore essential for consumer protection. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid and sensitive detection of lupin DNA. Several nuclear and chloroplast regions were evaluated for primer design, and gene encoding the Lup a 1 allergen was selected as the optimal target. Amplification was monitored by real-time fluorescence, agarose gel electrophoresis, and visual colorimetry. The selected primer set achieved a detection limit of 25 pg of lupin DNA and consistently detected lupin in binary mixtures down to 10 mg/kg, with no cross-reactivity against closely related legumes or tree nuts. Application to processed foods confirmed detection in products declaring lupin and revealed potential undeclared presence in some commercial samples. Colorimetric detection provided reliable results comparable to real-time monitoring, enabling simple readouts without specialized equipment. Overall, the developed LAMP assay represents a rapid, specific, and sensitive alternative to PCR-based methods for allergen monitoring and food safety management. Full article

Errors in de novo synthesized DNA can originate from the oligonucleotides used during assembly. Oligonucleotides may contain substitutions, deletions, and insertions resulting from either incomplete reactions at individual steps of the phosphoramidite synthetic cycle or various side reactions. In this study, we quantitatively assessed errors in both gene constructs assembled from synthetic oligonucleotides by Sanger sequencing and in synthetic oligonucleotides by NGS. Our data demonstrate that side reactions involving carboxylic acid anhydrides during the capping step of oligonucleotide synthesis lead to the modification of guanine residues. This guanine modification subsequently results in the accumulation of G to A substitutions in the final gene constructs. We show that the error rate can be reduced by replacing the standard acetic anhydride-based capping mixture with anhydrides of carboxylic acids weaker than acetic acid. Furthermore, a more significant reduction in errors is achievable by using capping reagents based on phosphoramidite chemistry. Full article

Polydeoxyribonucleotide (PDRN), a DNA fragment mixture, exerts biological effects via adenosine A2A receptor and salvage pathway activation. Here, Paeonia lactiflora-derived PDRN (Peony PDRN) is proposed as a plant-based alternative to salmon-derived PDRN. While P. lactiflora is known for its medicinal properties, the biological functions of Peony PDRN have not been characterized. To validate and optimize its efficacy, we systematically compared the biological activities of three molecular weight groups of Peony PDRN (high, medium, and low) using in vitro assays and clinical studies. The low-molecular-weight fraction (Low-Peony PDRN) markedly enhanced skin cell proliferation and migration, upregulated extracellular matrix-related genes (COL1A1, COL5A1, ELN, and FBN1), and promoted keratinocyte differentiation and epidermal barrier formation by increasing COL7A1, IVL, FLG, and OCLN expression. It also reduced reactive oxygen species levels and suppressed key inflammatory mediators. Clinically, topical application of Low-Peony PDRN for 2 weeks markedly reduced transepidermal water loss in a sodium lauryl sulfate-induced skin damage model, enhancing barrier recovery (n = 10). Periorbital skin elasticity improved after 4 weeks of treatment (Approval No. Intertek IRB-202505-HR(1)-0001, 20 June 2025). These results indicate that Low-Peony PDRN is a promising plant-derived biomaterial of pharmacological and cosmetic significance, with potential to address skin aging. Full article

Intra-tumor heterogeneity (ITH) is a compounding factor for cancer prognoses and treatment. Single-cell DNA sequencing (scDNA-seq) provides cellular resolution of the variations in a cell and has been widely used to study cancer progression and the responses to drugs and treatments. While low-coverage scDNA-seq technologies typically provide a large number of cells, accurate cell clustering is essential for effectively characterizing the ITH. The existing cell clustering methods are typically based on either single-nucleotide variations (SNV) or copy number alterations (CNA), without leveraging both signals together. Since both SNVs and CNAs are indicative of cell subclonality, in this paper, we designed a robust cell-clustering tool that integrates both signals using a graph autoencoder. Our model co-trains the graph autoencoder and a graph convolutional network (GCN) to guarantee meaningful clustering results and to prevent all cells from collapsing into a single cluster. Given the low-dimensional embedding generated by the autoencoder, we adopted a Gaussian mixture model (GMM) to further cluster the cells. We evaluated our method on eight simulated datasets and a real cancer sample. Our results demonstrate that our method consistently achieved higher V-measure scores compared to SBMClone, an SNV-based method, and a K-means method that relies solely on CNA signals. These findings highlight the advantage of integrating both SNV and CNA signals within a graph autoencoder framework for accurate cell clustering. Full article

Cellular senescence is defined as a state of permanent cell cycle arrest, providing a natural barrier against cancer. However, senescent cells are very metabolically active and secrete a complex mixture of bioactive molecules collectively known as the senescence-associated secretory phenotype (SASP), which play a dual role in cancer biology. While the SASP can suppress tumors by facilitating immunosurveillance, it can also promote tumor progression by fostering a pro-inflammatory milieu, stimulating angiogenesis, enhancing invasiveness, and enabling immune evasion. In Head and Neck Cancers (HNCs), a highly heterogeneous group of malignancies, SASP has emerged as a critical player in disease progression and treatment resistance. Persistent DNA damage response (DDR) signaling drives SASP and thereby contributes to the progression of head and neck cancer by modulating the tumour microenvironment. It influences the tumor microenvironment (TME) by facilitating epithelial-to-mesenchymal transition (EMT), promoting cancer stem cell-like properties, and impairing the efficacy of radiotherapy, chemotherapy, and immune checkpoint inhibitors. These effects underscore the need for targeted interventions to regulate SASP activity. This review presents a comprehensive overview of the molecular mechanisms underlying SASP generation and its effects on HNCs. We discuss the dual roles of SASP in tumor suppression and progression, its contribution to therapy resistance, and emerging therapeutic strategies, including novel senolytic and senomorphic drugs. Finally, we highlight key challenges and future directions for translating SASP-targeted therapies into clinical practice, emphasizing the need for biomarker discovery, and a deeper understanding of SASP heterogeneity. By targeting the SASP, there is potential to enhance therapeutic outcomes and improve the management of HNCs. Full article

Background/Objectives: The sequencing of mitochondrial DNA is a valuable tool in forensic genetics, particularly in cases involving degraded samples or those with low nuclear DNA content. In this study, we performed an internal validation for an NGS-based typing of the mitochondrial DNA control region using the Precision ID mtDNA Control Region Panel on the Ion S5^TM sequencer (Thermo Fisher Scientific, Waltham, MA, USA). This validation enhances the scientific robustness, reliability, and judicial admissibility of the results in forensic cases. Methods: Six parameters were evaluated: minimum read depth, sensitivity, repeatability, concordance with Sanger, reproducibility and heteroplasmy detection employing ten negative controls, nine reference samples, a bone sample, and six experimental mixtures. Libraries were prepared using the Ion Chef^TM system, quantified on the Quantstudio^TM 5 Real-Time PCR, sequenced on the Ion GeneStudio^TM S5, and analyzed with Converge^TM software. Results: In this study, we found that a read depth threshold of 100 reads per position, an optimal concentration of 20 pg/µL, and a detection threshold of heteroplasmies of 20% are appropriate to obtain reliable genetic profiles. This supports the application of this method in forensic casework, in which initial concentrations may be around the optimal concentration exposed here due to the provenience of the samples. Conclusions: The results indicate that the NGS platform is suitable for forensic mtDNA analysis, even under low-template conditions, and offers higher sensitivity compared to Sanger sequencing. However, some limitations were observed in the coverage of specific amplicons, the detections of polymorphisms in homopolymeric regions, and in the detection of low-level heteroplasmies. Full article

Calcium phosphate cements (CPCs) and biomaterials, such as mesoporous bioactive glass (MBG), are critical for bone tissue engineering. This study aimed to 3D-print CPC scaffolds modified with MBG to enhance their osteogenic potential and regenerative ability. MBG powder was synthesized and characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), and nitrogen adsorption–desorption techniques. A commercial CPC ink (hydroxyapatite/α-tricalcium phosphate) was mixed with 5% MBG (w/w; CPC/MBG), and, after rheological assessment, the mixture was used to obtain scaffolds via 3D printing. These scaffolds were then tested for chemical, morphological, and mechanical properties, as well as ion release analysis. Unmodified CPC 3D-printed scaffolds served as controls. Biological experiments, including cell viability, DNA content, cell adhesion/spreading, and osteogenic gene expression, were performed by seeding alveolar bone-derived mesenchymal stem cells onto the scaffolds. Statistics were performed using Student’s t-test and ANOVA with post hoc tests (α = 5%). MBG characterization showed a typical mesoporous structure with aligned microchannels and an amorphous structure. Both formulations released calcium and phosphate ions; however, CPC/MBG also released silicon. Cell viability, adhesion/spreading, and DNA content were significantly greater in CPC/MBG scaffolds compared to CPC (p < 0.05) after 3 and 7 days of culture. Furthermore, CPC/MBG supported increased expression of key osteogenic genes, including collagen (COL1A1), osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2), after 14 days (p < 0.05). The combination of CPC ink with MBG particles effectively enhances the biocompatibility and osteogenic potential of the scaffold, making it an innovative bioceramic ink formulation for 3D printing personalized scaffolds for bone regeneration. Full article

Cytostatics are contaminants of emerging concern. Their increasing presence in waste- and surface water is becoming a risk to aquatic life. Among them, 5-fluorouracil (5-FU) is one of the most frequently prescribed cytostatic drugs. 5-FU inhibits the thymidylate synthase activity, causing the depletion of thymidine nucleotides and misincorporation of uracil, and thus blocks DNA synthesis and replication. This study focuses on the influence of 5-FU on brackish and marine diatoms from the Baltic Sea, including Bacillaria cf. paxillifera, Gedaniella sp., Navicula perminuta, Nitzschia cf. aurariae, Skeletonema marinoi and Stephanocyclus meneghinianus, as well as natural microphytobenthos assemblages. The toxic effects of 5-FU were investigated in acute growth inhibition tests, which were performed using four types of media, i.e., artificial seawater with a salinity of 6.7, natural Baltic water, artificial seawater with a salinity of 22, and artificial seawater with the addition of cyclophosphamide and ifosfamide. The toxicity of 5-FU was checked for (1) each strain grown individually in all media, (2) six-strain mixed cultures grown in artificial seawater, and (3) natural microphytobenthic communities maintained in natural Baltic water. The diatom responses to 5-FU were species-specific. Growth conditions significantly modified the toxicity of 5-FU; tested strains were the most resistant to 5-FU when grown under optimal conditions, i.e., in natural Baltic water and/or at the optimal salinity. In the six-strain mixed cultures, higher 5-FU concentrations (>0.1 mg L⁻¹) shifted the dominance of diatom strains; the most resilient diatom S. meneghinianus replaced two other fast-growing strains, i.e., B. cf. paxillifera and Gedaniella sp. In the tested microphytobenthos assemblages, the highest biomass and species diversity were observed under the highest 5-FU concentrations (>5 mg L⁻¹). This indicated that the responses of complex species mixtures were governed by the ecophysiological features of their members and interactions among them, shaping the adaptive capacity of the entire assemblage. The introduction of the ecophysiological approach to toxicity testing seems to be crucial, and it would enable more realistic environmental risk assessment. Full article

Adeno-associated viruses (AAVs) are a leading vector for gene therapy, yet their clinical utility is limited by the lack of robust quality control methods to distinguish between empty (AAV_empty), partially loaded (AAV_partial), and fully DNA loaded (AAV_full) capsids. Current analytical techniques provide partial insights but remain limited in sensitivity, throughput, or resolution. Here we present a multimodal plasmonic nanopore sensor that integrates optical trapping with electrical resistive-pulse sensing to characterize AAV9 capsids at the single-particle level in tens of μL sample volumes and fM range concentrations. As a model system, we employed AAV9 capsids not loaded with DNA, capsids loaded with a self-complementary 4.7 kbp DNA (AAV_scDNA), and ones loaded with single-stranded 4.7 kbp DNA (AAV_ssDNA). Ground-truth validation was performed with analytical ultracentrifugation (AUC). Nanosensor data were acquired concurrently for optical step changes (occurring at AAV trapping and un-trapping) both in transmittance and reflectance geometries, and electrical nanopore resistive pulse signatures, making for a total of five data dimensions. The acquired data was then filtered and clustered by Gaussian mixture models (GMMs), accompanied by spectral clustering stability analysis, to successfully separate between AAV species based on their DNA load status (AAV_empty, AAV_partial, AAV_full) and DNA load type (AAV_scDNA versus AAV_ssDNA). The motivation for quantifying the AAV_empty and AAV_partial population fractions is that they reduce treatment efficacy and increase immunogenicity. Likewise, the motivation to identify AAV_scDNA population fractions is that these have much higher transfection rates. Importantly, the results showed that the nanosensor could differentiate between AAV_scDNA and AAV_ssDNA despite their identical masses. In contrast, AUC could not differentiate between AAV_scDNA and AAV_ssDNA. An equimolar mixture of AAV_scDNA, AAV_ssDNA and AAV_empty was also measured with the sensor, and the results showed the expected population fractions, supporting the capacity of the method to differentiate AAV load status in heterogeneous solutions. In addition, less common optical and electrical signal signatures were identified in the acquired data, which were attributed to debris, rapid entry re-entry to the optical trap, or weak optical trap exits, representing critical artifacts to recognize for correct interpretation of the data. Together, these findings establish plasmonic nanopore sensing as a promising platform for quantifying AAV DNA loading status and genome type with the potential to extend ultra-sensitive single-particle characterization beyond the capabilities of existing methods. Full article

Characterizing the botanical composition of pollen is essential to understanding the floral resources used by bees. While microscopy is the traditional method, it is time-consuming and limited in taxonomic resolution. Molecular tools such as DNA barcoding offer a more precise and cost-effective alternative for identifying plant taxa in mixed pollen samples. This study implemented a preliminary and cost-effective molecular approach to identify the botanical origin of pollen stored in bee bread from Apis mellifera hives in a tropical dry forest fragment in La Paz, Cesar, using rbcL and matK genes as markers. The chloroplast markers rbcL and matK were amplified and Sanger-sequenced from three independent bee hives, each processed in duplicate as technical replicates. The BLAST+ 2.17.0 results from Sanger sequences showed a sequence identity ranging from 89%–99%, with rbcL showing higher and more consistent matches than matK, suggesting stronger discriminatory power, while the lower identity in one hive indicated a more complex pollen mixture. However, matK detected a greater number of taxa overall (i.e., 70% of the total, 64 genera) compared with rbcL (i.e., 50%, 46 genera). Both markers overlapped in approximately 20% of the taxa, most of which (i.e., 94%) belonged to the family Cactaceae. This indicated that, although rbcL provided more reliable matches, matK contributed to broader taxonomic coverage, highlighting the complementarity of both markers for mixed pollen analyses. This approach highlights its value as an exploratory tool prior to applying high-throughput sequencing strategies. Furthermore, such studies may support the development of local honey brands by validating that their products originate mainly from the biodiversity of tropical dry forests, an ecosystem currently at risk, thereby conferring both ecological and market value. Full article

Radioprotective agents derived from natural food sources represent promising candidates for reducing the harmful effects of ionizing radiation and supporting healthy aging. In this study, we investigated the effects of selected micronized bioactive compounds and their mixes on DNA damage response pathways in human retinal epithelial cells (hTERT-RPE1). Individual compounds and their combinations were applied to cultured cells, and the expression of IER5, a radiation-inducible gene associated with DNA repair and cell survival, was evaluated, showing that most potent compound to be lycopene and quercetin. Thus, in the next step, commonly consumed foods available on the Czech market rich in moth—tomato and garlic—were analyzed for their antioxidant capacity. The results revealed marked variability in antioxidant potential among food sources, with specific cultivars exhibiting significantly higher values. Importantly, experimental mixtures of pure and micronized compounds demonstrated distinct and sometimes opposing effects on IER5 expression. These findings indicate that the radioprotective activity of dietary antioxidants depends not only on the properties of individual compounds but also on their specific combinations. Our study provides evidence that phytochemicals such as quercetin, lycopene, but also partially resveratrol and curcumin can modulate DNA-repair-associated pathways and underscores their potential as combinatory agents in strategies aimed at promoting genomic stability and potentially healthy aging. Full article

Photocatalytic oxidation of microorganisms is a powerful alternative to established disinfection approaches, applicable to a variety of water matrices. Bacterial vegetative cells, spores, fungi, and viruses, represent potential biopathogens and photocatalysis targets. Inactivation efficiency is usually evaluated by assessing viability through culture. However, additional inactivation assessment approaches are needed, as some microbes, despite being unculturable, remain metabolically active and pathogenic. Nucleic acid quantification approaches (qPCR) can assess nucleic acid release and degradation during photocatalysis. We developed a novel multiplex qPCR assay for simultaneous detection/quantification of genomic DNA from different bacterial and fungal species and of MS2 bacteriophage load. Following small-scale solar titanium dioxide photocatalysis on a microbial suspension mixture containing different biopathogen classes, we assessed photocatalytic efficiency by conventional microbiological assays (culture) and our novel molecular assay. Microbiological assays show a significant reduction in microbe viability within one hour of processing, following previously reported patterns of microbial species resistance. Molecular analysis data show that nucleic acids released in solution due to microbial oxidative damage were significantly reduced due to oxidative degradation within six hours. Through targeting different biopathogen classes, our assay could be a useful tool for assessment of photocatalytic microbe inactivation both in laboratory and real-wastewater applications. Full article