Search Results (128)

Aptamers are powerful tools for detecting and analyzing biomolecules that consist of proteins or nucleic acids. However, their application to aptamers against viroids—highly structured self-replicating RNAs—has not yet been explored. In this study, a magnetic bead- and high-throughput sequencing-based SELEX (MB-HTS-SELEX) protocol for selecting potential aptamers against potato spindle tuber viroid (PSTVd) is presented. Full-length biotinylated-PSTVd RNA was transcribed in vitro, immobilized on streptavidin-coated magnetic beads, and incubated with a library of ~3.32 × 10¹⁴ molecules of random single-stranded oligo-DNAs (oligo-ssDNAs) of 20, 30, or 40 nucleotides (L20, L30, or L40, respectively) flanked by primer binding sites for downstream PCR amplification. Simultaneous biotin labeling of the anti-aptamer strand of the resulting double-stranded DNA (dsDNA) amplicons facilitated strand separation using streptavidin-coated magnetic beads. After 10 selection rounds, high-throughput sequencing, followed by bioinformatics analysis of the generated sequences, allowed for the detection of several enriched sequences, representing putative PSTVd-binding aptamers. Subsequent pull-down assays showed that the most abundant oligo-ssDNA in L30 was docked on PSTVd molecules. This combination method may ameliorate the selection of high-affinity aptamers against PSTVd, reduce the number of selection cycles, time, and other costs of aptamer production, thereby promoting future massive and cost-effective viroid detection and characterization. Full article

Background: Hypoxia is a key driver of cancer progression. However, its specific prognostic significance in gastric cancer (GC) remains insufficiently characterized. Methods: Single-sample gene set enrichment analysis (ssGSEA), weighted gene co-expression network analysis (WGCNA), univariate Cox regression, and least absolute shrinkage and selection operator (LASSO) regression were employed to identify a hypoxia-related prognostic signature. Subsequently, immune microenvironment profiling and single-cell RNA sequencing analyses were employed to further characterize the biological characteristics of the signature. In addition, quantitative real-time polymerase chain reaction (qPCR) was used to validate the expression levels of key hypoxia-associated genes in human GC tissues. Results: Elevated hypoxia levels were linked to worse survival outcomes in GC patients. Through integrated WGCNA, Cox, and LASSO analyses, a hypoxia-related prognostic signature (HYS) consisting of four genes—SPARC, AXL, NRP1, and VCAN—was established. Patients in the HYS-high group exhibited markedly poorer overall survival than their HYS-low counterparts [p = 0.000126, hazard ratio (HR) = 1.936]. Moreover, the HYS-high group exhibited increased infiltration of resting CD4⁺ memory T cells, monocytes, M2 macrophages, and resting mast cells, as well as elevated expression of immunosuppressive molecules, including PDCD1LG2 and HAVCR2. Single-cell RNA sequencing analysis revealed that the signature genes were predominantly expressed in cancer-associated fibroblasts. Consistently, qPCR analysis in five paired GC and para-carcinoma tissues confirmed higher expression of these genes in tumor samples (p < 0.01). Conclusions: Our findings indicate that hypoxia is a critical determinant of prognosis in GC and is closely associated with an immunosuppressive tumor microenvironment, highlighting its potential value as a prognostic biomarker and therapeutic target. Full article

Cell adhesion molecules (CAMs) are key regulators of tissue structural integrity and functional coordination, yet their specific role in the adaptation of yak lung tissue to high-altitude hypoxia remains unelucidated. Thus, we employed transcriptomic sequencing (RNA-seq), molecular biology assays, and single-cell RNA-seq (scRNA-seq) to analyze the expression characteristics of CAMs in yak lung tissues at high and low altitudes. Trypsin or collagenase digestion showed higher cell counts in high-altitude yak lungs (p < 0.05). RNA-seq analysis revealed significant enrichment of differentially expressed genes (DEGs) in adhesion-related pathways. Inductively coupled plasma mass spectrometry detected elevated Ca²⁺ levels in high-altitude yak lungs (p < 0.05). Quantitative real-time PCR (qRT-PCR) detection of key genes from five major families of CAMs revealed the downregulation of cadherin and integrin family-related genes, and upregulation of immunoglobulin superfamily-related genes, in high-altitude yak lungs (p < 0.05), corroborated by immunohistochemical (IHC) staining. A 10× scRNA-seq revealed adhesion changes in 9 of 15 lung cell subpopulations, with differentially expressed CAMs involving integrins. This study demonstrates that yak lung tissue establishes a sophisticated adhesive homeostasis through differential CAMs regulation. This strategy optimizes pulmonary immune responses and energy allocation, ensures structural integrity and functional coordination, and thereby facilitates superior acclimatization to higher-altitude hypoxia. Full article

Mesenchymal stromal cells (MSCs) are multipotent and play an important role in regenerative processes such as wound healing. Data on possible changes and functional restrictions of MSCs due to cisplatin chemotherapy, for example, in the treatment of head and neck cancer, diverge. The aim of this study was to evaluate the influence of cisplatin on MSCs with regard to their defining characteristics and their ability to differentiate and to migrate. MSCs from four human donors (a 59-year-old man, a 63-year-old woman, a 70-year-old man, and a 61-year-old man) were cultured in vitro with and without cisplatin for 24 h, and toxic and subcytotoxic concentrations were determined using an MTT. We then examined the surface phenotype markers (flow cytometry), migration (scratch assay), histological differentiation markers (adipo-, chondro-, osteogenic), and the expression of selected line-associated genes in real-time quantitative PCR (RT-qPCR) (LEP, SOX9, RUNX2). These characteristics were evaluated after treatment with different subcytotoxic, clinically relevant doses of cisplatin. Flow cytometry confirmed the presence of MSCs-characteristic surface markers, which remained stable under treatment with subcytotoxic doses of cisplatin. Cisplatin exposure reduced the mRNA abundance of leptin (a marker for adipogenic differentiation) but increased SOX9 mRNA abundance (chondrogenic differentiation). RUNX (osteogenic differentiation) did not change post cisplatin exposure. Histological analysis showed no difference with regard to osteogenic, chondrogenic, and adipogenic differentiation at doses up to 10 μM cisplatin. Cell migration was not restricted by cisplatin exposure under the conditions used here. The characteristics of MSCs were not different to controls post cisplatin exposure. mRNA analysis suggested induced changes by cisplatin, although this effect was not histologically detectable even at high doses. Based on the single-molecule markers used here, indications for an inhibitory effect of cisplatin on adipogenic differentiation and a rather enhancing effect on chondrogenic and osteogenic differentiation may be hypothesized. The process observed here could further aggravate the already serious problem of malnutrition in head and neck cancer patients, for example. Taken together though, our study confirms overall MSCs tolerance towards cisplatin. Full article

RNA modifications are essential regulators of gene expression and cellular function, modulating RNA stability, splicing, translation, and localization. Dysregulation of these modifications has been linked to cancer, neurodegenerative disorders, viral infections, and other diseases. Precise quantification and mapping of RNA modifications are crucial for understanding their biological roles. This review summarizes current and emerging methodologies for RNA modification analysis, including mass spectrometry, antibody-based and non-antibody-based approaches, PCR- and NMR-based detection, chemical- and enzyme-assisted sequencing, and nanopore direct RNA sequencing. We also highlight advanced techniques for single-cell and single-molecule imaging, enabling the study of modification dynamics and cellular heterogeneity. The advantages, limitations, and challenges of each method are discussed, providing a framework for selecting appropriate analytical strategies. Future perspectives emphasize high-throughput, multiplexed, and single-cell approaches, integrating multiple technologies to decode the epitranscriptome. These approaches form a robust toolkit for uncovering RNA modification functions, discovering biomarkers, and developing novel therapeutic strategies. Full article

Objectives: Fibroblast growth factor 9 (FGF9), a crucial member of the FGF family, functions as an intercellular signaling molecule involved in angiogenesis, embryogenesis, and tissue repair. Our previous study demonstrated that FGF9 expression in chicken hierarchical granulosa cells (Post-GCs) is regulated by LSD1 Ser54 phosphorylation and that FGF9 promotes cell proliferation. This study aims to analyze the expression and regulation of the FGF9 gene in chicken ovarian follicles and its genetic effect on laying traits in hens. Methods: Chicken FGF9 mRNA expression patterns were examined by real-time quantitative PCR (RT-qPCR). Detection of single nucleotide polymorphisms (SNPs) was performed using PCR amplification and Sanger sequencing. Transcription activity was compared using dual-luciferase reporter assay. Results: Following follicle selection, chicken FGF9 expression significantly decreased in granulosa cells (p < 0.05) while it increased in theca cells (p < 0.05). Hormonal treatments revealed differential regulation; estradiol and FSH downregulated FGF9 in both pre-hierarchical and hierarchical granulosa cells (p < 0.05), whereas progesterone exhibited opposing effects, suppressing expression in pre-hierarchical granulosa cells (Pre-GCs) but stimulating its expression in Post-GCs (p < 0.05). In theca cells, estradiol consistently inhibited FGF9 expression (p < 0.05), while FSH only affected FGF9 expression in pre-hierarchical follicles. Six SNPs in the promoter region (g.−1965G>A, g.−2177G>A, g.−2289G>A, g.−3669A>G, g.−3770A>G, g.−3906G>A) were identified, five of which (g.−1965G>A, g.−2177G>A, g.−2289G>A, g.−3669A>G, g.−3906G>A) showed significant associations with egg production traits. Notably, alleles A (g.−2289), G (g.−3669), and A (g.−3906) enhanced the transcription activity of chicken FGF9 in Pre-GCs. Conclusions: These findings provide novel insights into the expression pattern and regulatory mechanisms of chicken FGF9 during follicular development and identify some genetic markers for egg-laying traits in chickens. Full article

Background: Diabetic foot ulcers (DFUs) are chronic wounds with high morbidity and mortality. Secretory proteins coordinate intercellular communication and may regulate inflammation, tissue repair and regeneration, but their contributions to DFU pathogenesis remain unclear. Aim: To discover and validate secretory protein–linked biomarkers that distinguish non-healing DFUs and to explore their potential utility for diagnosis and therapy. Methods: We integrated bulk RNA-sequencing datasets (GSE199939 training set; GSE80178 and GSE143735 validation sets) and a single-cell RNA-sequencing dataset (GSE223964). Differentially expressed genes, secretory protein lists, and weighted gene co-expression networks were intersected to select candidates. Functional enrichment, protein interaction networks and support vector machine–recursive feature elimination identified key markers. We visualized their cell-type distribution at single-cell resolution and validated their expression in external cohorts. Pathway enrichment, gene co-expression networks, ceRNA regulatory analysis and qRT-PCR in patient samples were used for further characterization. Results: Among 4803 differentially expressed genes, 743 overlapped with known secretory proteins. WGCNA highlighted modules strongly associated with DFUs, yielding 386 candidates. SVM-RFE combined with protein interaction analysis pinpointed four secretory proteins—LYZ, MPO, SLCO2B1 and TREM2—as putative biomarkers. Single-cell analyses showed that MPO, LYZ, SLCO2B1 and TREM2 transcripts are detectable in multiple skin-resident and immune cell populations, and that the DFU-associated upregulation of MPO and LYZ is most pronounced within keratinocyte clusters, while MPO and TREM2 remained consistently dysregulated in independent bulk cohorts. MPO-associated genes were enriched for immune and inflammatory pathways, whereas TREM2-linked genes implicated cell cycle and cytoskeletal regulation. GeneMANIA and ceRNA analyses revealed extensive interaction networks. qRT-PCR confirmed differential expression of MPO and TREM2 in clinical DFU tissues. Conclusions: Integrated multi-modal analysis identifies secretory proteins, particularly MPO and TREM2, as central determinants of impaired healing in DFUs. These molecules and their regulatory networks represent promising biomarkers and therapeutic targets for precision management of diabetic wounds. Full article

Background: The emergence of the COVID-19 pandemic has accelerated research into diverse immune response mechanisms. One key area of interest is the regulation of cytotoxic activity by Natural Killer (NK) cells. These cells rely on a dynamic interplay between activating and inhibitory surface receptors that recognize specific ligands on target cells. Among these, receptors from the NKG2 family are particularly important, as maintaining their proper balance and function is essential for controlling NK cell cytotoxicity. Methods: In this study we employed qPCR to assess the genetic variability using single-nucleotide polymorphisms (SNPs) of NKG2A and NKG2D receptors and their ligands HLA-E and MICA/MICB. NKG2C deletion was determined by PCR-SSP, and serum-soluble levels of HLA-E and MICA/MICB molecules were measured by ELISA and Luminex methods. Results: Genotyping studies revealed that both NKG2A rs7301582 T and HLA-E rs1264457 A (HLA-E*01:01) alleles were predominant among infected individuals (OR = 2.21, p = 0.0258 and OR = 2.84, p = 0.0257, respectively). In contrast to MICB rs1065075 A, the MICA rs1051792 A (129Met) allele was most commonly found in hospitalized patients (OR = 14.95, p = 0.0114). The presence of the NKG2C del variant tended to be associated with an increased risk of SARS-CoV-2 infection (OR = 2.02, p = 0.0694). Moreover, higher concentrations of serum-soluble MICB was detected in infected individuals as compared to the control group (p = 0.008). Conclusions: Genetic variability of NK cell receptors and ligands as well as serum levels of their soluble forms showed associations with the risk of development of COVID-19 and the severity of its symptoms. Full article

Background/Objectives: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a well-established, minimally invasive method for sampling mediastinal and hilar lymph nodes. It has high sensitivity and specificity for diagnosing lymph node involvement. However, achieving a definitive cytopathological diagnosis can be challenging owing to the limited presence of atypical tumor cells. The scanning single-molecule counting (SSMC) method enables rapid measurement of target molecule expression. This study assessed the correlation between miR-200c expression measured by SSMC and lymph node metastasis confirmed by cytopathology. Methods: Following EBUS-TBNA in patients with lung cancer or suspected lung cancer, we flushed 22-gauge biopsy needles with 1 mL saline and extracted microRNA (miRNA) from the lavage fluid. The quality of SSMC results for miR-200c was evaluated and compared with that of quantitative real-time polymerase chain reaction (RT-qPCR). Results: Linear regression analysis of miR-200c between SSMC and RT-qPCR showed a statistically significant positive correlation (R² = 0.81, p < 0.0001), demonstrating the feasibility of SSMC for evaluating miRNA in lymph nodes. Based on these findings, we enrolled and analyzed 100 lymph nodes from 86 patients. High miR-200c expression detected lymph node metastasis with high sensitivity (85.7%) and specificity (83.3%) and an AUC of 0.88. No discordance was observed between the standard SSMC and rapid methods. Conclusions: The expression of miR-200c, as measured by SSMC, may serve as a biomarker for molecular nodal staging. The rapid SSMC method provides results within 30 min, significantly faster than conventional RT-qPCR, and may complement rapid on-site cytological evaluation. Full article

There have been rising interests in ultra-sensitive biosensing technologies for early diagnosis and prognosis monitoring of infectious diseases, cancers, and neurodegenerative diseases. Digital signal readout strategy represented by digital ELISA or digital PCR, advanced biosensing field enormously, which enables detection of biomolecules under the detection limit of conventional biosensing methods. However, due to the need for compartmentalization and limited multiplex capability, it has been hurdled for utilization in applications requiring hierarchical resolution analysis such as sub-cellular molecules or molecular cargo of single cells or single extracellular vesicles (EVs). Rolling circle amplification (RCA), an isothermal DNA amplification method enabling localization of an amplified signal, can eliminate the need for compartmentalization and increase multiplex capability. It also has potential to expand applications of single molecule counting assay for understanding hierarchy of biological systems. In this review, recent advances in RCA-based single molecule counting assay are overviewed and their applications in single cells and single EVs quantitative analysis are discussed. Furthermore, the limitations and outlook of RCA-based single molecule counting assay are highlighted. Full article

Digital PCR, as a nucleic acid absolute quantification method at the single-molecule level, has been widely applied in early cancer screening, single-cell analysis, and other biomedical fields. However, existing digital PCR methods still suffer from high costs, complex operations, and low detection dynamic range, which limit their applications. In the study, we developed a microfluidic chip-based digital PCR with a high-density vertical structure using PDMS (polydimethylsiloxane) flexible material. The chip features a three-layer structure of glass–PDMS–glass, with the PDMS structural layer containing 30,000 reaction chambers, each with a volume of 0.713 nL. This vertical-structured chip can increase the total volume and the total number of chambers by 50% without changing the chip area and chamber volume, thereby significantly enhancing dynamic range and sensitivity of the chip detection. This chip is theoretically capable of achieving a nucleic acid detection dynamic range close to 10⁵. Moreover, the digital PCR quantitative detection results of five different concentrations of serially diluted KRAS plasmid DNA templates using this chip also validated the accuracy and reliability of the nucleic acid quantitative detection results. The vertical-structured digital PCR chip, with its simple manufacturing process, uniform and stable sample partitioning, wide detection dynamic range, and low cost, will promote the widespread application of digital PCR. Full article

Background and Objectives: A thorough comprehension of the essential molecules and related processes underlying the carcinogenesis, proliferation, and recurrence of acute myeloid leukemia (AML) is crucial. This study aimed to investigate the expression levels, diagnostic and prognostic significance and biological roles of Bcl-2-associated athanogene 4 (BAG4) in AML carcinogenesis. Materials and Methods: Gene expression profiles were analyzed using publicly available datasets, particularly GSE9476 and TCGA, using tools such as GEO2R, GEPIA2, UALCAN and TIMER2.0. The immune infiltration correlation was examined using the GSCA platform, while the function of BAG4 at the single-cell level was analyzed via CancerSEA. Protein–protein and gene–gene interaction networks were constructed using STRING and GeneMANIA, and enrichment analyses were performed using GO, KEGG and DAVID. Expression validation was performed using RT-qPCR in HL-60 (AML) and HaCaT (normal) cells, and ROC curve analysis evaluated the diagnostic accuracy. Results: BAG4 was significantly overexpressed in AML tissues and cell lines compared with healthy controls. High BAG4 expression was associated with poor overall survival and strong diagnostic power (AUC = 0.944). BAG4 was positively associated with immune cell infiltration and negatively associated with CD4+/CD8+ T and NK cells. At the single-cell level, BAG4 was associated with proliferation, invasion, and DNA repair functions. Functional network analysis showed that BAG4 interacted with apoptosis and necroptosis-related genes such as BCL2, BAG3 and TNFRSF1A and was enriched in pathways such as NF-κB, TNF signaling and apoptosis. Conclusions: BAG4 is overexpressed in AML and is associated with adverse clinical outcomes and immune modulation. It may play an important role in leukemogenesis by affecting apoptotic resistance and immune evasion. BAG4 has potential as a diagnostic biomarker and treatment target in AML, but further in vivo and clinical validation is needed. Full article

Mpox is a zoonotic disease caused by the Mpox virus (MPXV). The rapid and accurate diagnosis of MPXV is essential for the timely and effective prevention, control, and treatment of the disease. In this study, we combined Multienzyme Isothermal Rapid Amplification (MIRA) (at 42 °C) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 12b(CRISPR/Cas12b) (at 60 °C) to develop a single-tube two-step assay for rapid MPXV detection, leveraging the distinct physical states of tricosane at these temperatures. MIRA amplification primers and CRISPR/cas12b SgRNA were designed based on the MPXV F3L gene. After screening the primers and sgRNAs, the reaction conditions were optimized, and the performances of the assay were evaluated. The detection limit (LOD) of this single-tube two-step MIRA-CRISPR/Cas12b assay for MPXV is four copies of DNA molecules. No cross-reactivity with other pathogens (herpes simplex virus (HSV), Epstein–Barr virus (EBV), Coxsackievirus A16 (CVA16), Enterovirus A71 (EV-A71), and measles virus (MeV)) was found. The assay also showed good consistency with quantitative real-time PCR (qPCR) (Kappa = 0.9547, p < 0.05, n = 100) in the detection of clinical samples, with a sensitivity of 98.5% and a specificity of 97.0%. The single-tube two-step MIRA-CRISPR/Cas12b assay permits the rapid (within 45 min), sensitive, and specific detection of MPXV. The lack of need for opening the reaction tube eliminates the risk of product contamination. Full article

Cannabis sativa L. shows potent anti-inflammatory activity, resulting in an interesting pharmacological option for pain management. The aim of the study was to evaluate the association between pharmacogenetics, neurological and inflammatory biomarkers, and cannabinoid plasma exposure in patients treated with cannabis. A total of 58 patients with a diagnosis of neuropathic and chronic pain treated with medical cannabis were analyzed. Cannabis was administered as a decoction (n = 47) and as inhaled cannabis (n = 11): 30 patients were treated with cannabis with high THC, while 28 patients were treated with cannabis with reduced THC (plus CBD). Cannabinoid plasma concentrations were obtained with UHPLC-MS/MS. Allelic discrimination was assessed by real-time PCR. Inflammation biomarkers (e.g., interleukin-10) were analyzed by ELISA, neurofilaments light chain (NFL), and brain-derived neurotrophic factor (BDNF) by Single Molecule Array. A statistically significant difference in IL-10 (p = 0.009) and BDNF (p = 0.004) levels was observed comparing patients treated with decoction and inhaled cannabis. BDNF and NFL results correlated with cannabinoid concentrations. Concerning genetics, the COMT 680 T>C genetic variant influences cannabinoid plasma levels, including Δ9-THC (p = 0.017). Conclusions: This study shows a possible impact of some genetic variants on cannabinoid plasma exposure, other than a possible role of medical cannabis on inflammation-related and neuronal impairment factor levels. Further studies in larger cohorts are required. Full article

Salt-sensitive hypertension (SSH) is closely associated with arterial inflammation, yet its molecular mechanisms remain unclear. In this study, we utilized deoxycorticosterone acetate (DOCA)-salt-induced hypertensive mice, which exhibited elevated blood pressure and significant arterial inflammation. Single-cell RNA sequencing (scRNA-seq) identified interferon regulatory factor 5 (IRF5) and its downstream targets, signal transducer and activator of transcription (STAT), as key regulators of these inflammatory changes. In vivo, IRF5 levels were significantly elevated in the DOCA group, while STAT1 and STAT2 protein levels were comparable to those in the normal salt group. However, nuclear levels of phosphorylated STAT1 (pSTAT1) and phosphorylated STAT2 (pSTAT2) were markedly higher in the DOCA group. Furthermore, scRNA-seq analysis showed increased IRF5 expression in endothelial cells (ECs) in both human and mouse aorta samples. In vitro, IRF5 knockdown in artery ECs led to a reduction in nuclear pSTAT1 and pSTAT2 expression. These results suggest that IRF5 promotes STAT1 and STAT2 phosphorylation, enabling their nuclear translocation. Additionally, RNA sequencing indicated a positive correlation between endothelial cell-specific molecule 1 (ESM1) and STAT1/STAT2. Using the UCSC and JASPAR databases, we identified multiple binding sites for the STAT1::STAT2 dimer on the ESM1 promoter. Luciferase reporter assays revealed enhanced ESM1 transcription following pSTAT1::pSTAT2 binding, and pinpoint potential binding sites. Chromatin Immunoprecipitation Quantitative PCR (ChIP-qPCR) further confirmed the specific binding sites between the pSTAT1::pSTAT2 dimer and the ESM1 promoter. These findings highlight the critical role of the IRF5-pSTAT1::pSTAT2-ESM1 pathway in the pathogenesis of SSH and suggest potential therapeutic targets. Full article