Search Results (22)

The development of low-background, facile, and robust fluorescent nanoprobes for imaging and monitoring of intracellular mRNA changes remains a great challenge. Taking advantage of the high fluorescence quenching efficiency of core-shell gold@polydopamine (Au@PDA) nanocomposites and Ca²⁺-promoting DNA adsorption stability, a simple and universal bioconjugate strategy was designed to a construct fluorescent nanoprobe for highly efficient tumor-related mRNA imaging. The fluorescence of Cy5-labeled DNA was quenched up to 92.38% by the AuNP and PDA via nanometal surface energy transfer (NSET) and photoinduced electron transfer (PET), respectively. TK1 mRNA, a biomarker of tumor growth, initiates hybridization and results in fluorescence recovery, which built the foundation for identifying the expression level changes in living cells. More importantly, three kinds of tumor-related mRNA (TK1 mRNA, GalNAc-T mRNA, and C-myc mRNA) can be detected simultaneously with different fluorophore-modified recognition sequences, which can avoid false positive signals and improve the reliability of cancer diagnostic, holding great promise for cancer diagnosis, prognosis, and therapy. Full article

Multitarget drug discovery, including host-directed therapy, is particularly promising for tuberculosis (TB) due to the resilience of Mycobacterium tuberculosis (Mtb) as well as the complexity of the host’s immune response. In this proof-of-concept study, we used high-content imaging to test a novel panel of dual glycogen synthase kinase 3 beta (GSK-3β) and histone deacetylase (HDAC) 1 and 6 inhibitor candidates for their efficacy in reducing the growth of green fluorescent protein (GFP)-expressing mycobacteria in human primary macrophages. We demonstrate that all ten test compounds, also including the GSK-3β inhibitor SB415286, exhibit an antimycobacterial effect of 20–60% at low micromolar doses and are non-toxic to host cells. Mtb growth showed a positive correlation with the respective 50% inhibitory concentration (IC50) values of GSK-3β, HDAC1, and HDAC6 in each compound, indicating that compounds with a potent IC50 value for HDAC1, in particular, corresponded to higher antimycobacterial activity. Furthermore, the results from multiparametric flow cytometry and a customized multiplex RNA array demonstrated that SB415286 and selected compounds, C02 and C06, could modulate immune polarization and inflammation in Mtb-infected macrophages involving an enhanced expression of CCL2, IL-10 and S100A9, but a decrease in inflammatory mediators including COX-2, TNF-α, and NFκB. These data suggest that GSK-3β inhibition alone can decrease the intracellular growth of mycobacteria and regulate macrophage inflammation, while dual GSK-3β/HDAC inhibitors enhance this efficacy. Accordingly, the tailored design of dual GSK-3β/HDAC inhibitors could represent an innovative approach to host-directed therapy in TB. Full article

Ovarian cancer (OC), the seventh most common cancer in women and the most lethal gynecological malignancy, is a significant global health challenge, with >324,000 new cases and >200,000 deaths being reported annually. OC is characterized by late-stage diagnosis, a poor prognosis, and 5-year survival rates ranging from 93% (early stage) to 20% (advanced stage). Despite advances in genomics and proteomics, effective early-stage diagnostic tools and population-wide screening strategies remain elusive, contributing to high mortality rates. The complex pathogenesis of OC involves diverse histological subtypes and genetic predispositions, including BRCA1/2 mutations; notably, a considerable proportion of OC cases have a hereditary component. Current diagnostic modalities, including imaging techniques (transvaginal ultrasound, computed/positron emission tomography, and magnetic resonance imaging) and biomarkers (CA-125 and human epididymis protein 4), with varying degrees of sensitivity and specificity, have limited efficacy in detecting early-stage OC. Emerging technologies, such as liquid biopsy, multiomics, and artificial intelligence (AI)-assisted diagnostics, may enhance early detection. Liquid biopsies using circulating tumor DNA and microRNAs are popular minimally invasive diagnostic tools. Integrated multiomics has advanced biomarker discovery. AI algorithms have improved imaging interpretation and risk prediction. Novel screening methods including organoids and multiplex panels are being explored to overcome current diagnostic limitations. This review highlights the critical need for continued research and innovation to enhance early diagnosis, reduce mortality, and improve patient outcomes in OC and posits personalized medicine, integrated emerging technologies, and targeted global initiatives and collaborative efforts, which address care access disparities and promote cost-effective, scalable screening strategies, as potential tools to combat OC. Full article

The existence of cancer stem cells (CSCs) in various tumors has become increasingly clear in addition to their prominent role in therapy resistance, metastasis, and recurrence. For early diagnosis, disease progression monitoring, and targeting, there is a high demand for clinical-grade methods for quantitative measurement of CSCs from patient samples. Despite years of active research, standard measurement of CSCs has not yet reached clinical settings, especially in the case of solid tumors. This is because detecting this plastic heterogeneous population of cells is not straightforward. This review summarizes various techniques, highlighting their benefits and limitations in detecting CSCs from patient samples. In addition, methods designed to detect CSCs based on secreted and niche-associated signaling factors are reviewed. Spatial and single-cell methods for analyzing patient tumor tissues and noninvasive techniques such as liquid biopsy and in vivo imaging are discussed. Additionally, methods recently established in laboratories, preclinical studies, and clinical assays are covered. Finally, we discuss the characteristics of an ideal method as we look toward the future. Full article

Congenital titinopathies reported to date show autosomal recessive inheritance and are caused by a variety of genomic variants, most of them located in metatranscript (MTT)-only exons. The aim of this study was to describe additional patients and establish robust genotype–phenotype associations in titinopathies. This study involved analyzing molecular, clinical, pathological, and muscle imaging features in 20 patients who had at least one pathogenic or likely pathogenic TTN variant in MTT-only exons, with onset occurring antenatally or in the early postnatal stages. The 20 patients with recessive inheritance exhibited a heterogeneous range of phenotypes. These included fetal lethality, progressive weakness, cardiac or respiratory complications, hyper-CKemia, or dystrophic muscle biopsies. MRI revealed variable abnormalities in different muscles. All patients presented severe congenital myopathy at birth, characterized by arthrogryposis (either multiplex or axial–distal) or neonatal hypotonia in most cases. This study provides detailed genotype–phenotype correlations in congenital titinopathies caused by mutations in MTT-only exons. The findings highlight the variability in clinical presentation and the severity of phenotypes associated with these specific genetic alterations. RNA-seq analyses provided valuable insights into the molecular consequences of TTN variants, particularly in relation to splicing defects and nonsense-mediated RNA decay. In conclusion, this study reinforces the genotype–phenotype correlations between congenital myopathies and variants in TTN MTT-only exons, improves their molecular diagnosis, and provides a better understanding of their pathophysiology. Full article

The emergence of CRISPR/Cas systems has revolutionized the field of molecular diagnostics with their high specificity and sensitivity. This review provides a comprehensive overview of the principles and recent advancements in harnessing CRISPR/Cas systems for detecting DNA and RNA. Beginning with an exploration of the molecular mechanisms of key Cas proteins underpinning CRISPR/Cas systems, the review navigates the detection of both pathogenic and non-pathogenic nucleic acids, emphasizing the pivotal role of CRISPR in identifying diverse genetic materials. The discussion extends to the integration of CRISPR/Cas systems with various signal-readout techniques, including fluorescence, electrochemical, and colorimetric, as well as imaging and biosensing methods, highlighting their advantages and limitations in practical applications. Furthermore, a critical analysis of challenges in the field, such as target amplification, multiplexing, and quantitative detection, underscores areas requiring further refinement. Finally, the review concludes with insights into the future directions of CRISPR-based nucleic acid detection, emphasizing the potential of these systems to continue driving innovation in diagnostics, with broad implications for research, clinical practice, and biotechnology. Full article

GATA2 deficiency is a heterogeneous, multisystem disorder associated with a high risk of developing myelodysplastic syndrome (MDS) and the progression to acute myeloid leukemia. The mechanisms underlying malignant transformation in GATA2 deficiency remain poorly understood, necessitating predictive markers to assess an individual’s risk of progression and guide therapeutic decisions. In this study, we performed a systematic analysis of bone marrow biopsies from 57 pediatric MDS patients. Focusing on hematopoiesis and the hematopoietic niche, including its microenvironment, we used multiplex immunofluorescence combined with multispectral imaging, gene expression profiling, and multiplex RNA in situ hybridization. Patients with a GATA2 deficiency exhibited a dysregulated GATA2 transcriptional network. Disease progression (GATA2-EB, n = 6) was associated with increased GATA2 mRNA levels, restored expression of the GATA2 target EZH2, and increased H3K27me3. GATA2-EB was further characterized by the high expression of the anti-apoptotic protein BCL2, a feature absent in children with a GATA2 deficiency and refractory cytopenia of childhood (GATA2-RCC, n = 24) or other pediatric MDS subgroups (RCC, n = 17; MDS-EB, n = 10). The multispectral imaging analysis of additional BCL2 family members revealed significantly elevated Mediators of Apoptosis Combinatorial (MAC) scores in GATA2-EB patients. Taken together, our findings highlight the potential drivers of disease progression in GATA2 deficiency, particularly increased histone trimethylation and dysregulated apoptosis. Furthermore, upregulated BCL2 and EZH2 and increased MAC scores provide a strong rationale for the use of venetoclax and azacitidine in therapeutic regimens for GATA2-EB. Full article

This review summarizes recent advances in leveraging localized surface plasmon resonance (LSPR) nanotechnology for sensitive cancer biomarker detection. LSPR arising from noble metal nanoparticles under light excitation enables the enhancement of various optical techniques, including surface-enhanced Raman spectroscopy (SERS), dark-field microscopy (DFM), photothermal imaging, and photoacoustic imaging. Nanoparticle engineering strategies are discussed to optimize LSPR for maximum signal amplification. SERS utilizes electromagnetic enhancement from plasmonic nanostructures to boost inherently weak Raman signals, enabling single-molecule sensitivity for detecting proteins, nucleic acids, and exosomes. DFM visualizes LSPR nanoparticles based on scattered light color, allowing for the ultrasensitive detection of cancer cells, microRNAs, and proteins. Photothermal imaging employs LSPR nanoparticles as contrast agents that convert light to heat, producing thermal images that highlight cancerous tissues. Photoacoustic imaging detects ultrasonic waves generated by LSPR nanoparticle photothermal expansion for deep-tissue imaging. The multiplexing capabilities of LSPR techniques and integration with microfluidics and point-of-care devices are reviewed. Remaining challenges, such as toxicity, standardization, and clinical sample analysis, are examined. Overall, LSPR nanotechnology shows tremendous potential for advancing cancer screening, diagnosis, and treatment monitoring through the integration of nanoparticle engineering, optical techniques, and microscale device platforms. Full article

APOBEC3B (A3B) is aberrantly overexpressed in a subset of breast cancers, where it associates with advanced disease, poor prognosis, and treatment resistance, yet the causes of A3B dysregulation in breast cancer remain unclear. Here, A3B mRNA and protein expression levels were quantified in different cell lines and breast tumors and related to cell cycle markers using RT-qPCR and multiplex immunofluorescence imaging. The inducibility of A3B expression during the cell cycle was additionally addressed after cell cycle synchronization with multiple methods. First, we found that A3B protein levels within cell lines and tumors are heterogeneous and associate strongly with the proliferation marker Cyclin B1 characteristic of the G₂/M phase of the cell cycle. Second, in multiple breast cancer cell lines with high A3B, expression levels were observed to oscillate throughout the cell cycle and again associate with Cyclin B1. Third, induction of A3B expression is potently repressed throughout G₀/early G₁, likely by RB/E2F pathway effector proteins. Fourth, in cells with low A3B, induction of A3B through the PKC/ncNF-κB pathway occurs predominantly in actively proliferating cells and is largely absent in cells arrested in G₀. Altogether, these results support a model in which dysregulated A3B overexpression in breast cancer is the cumulative result of proliferation-associated relief from repression with concomitant pathway activation during the G₂/M phase of the cell cycle. Full article

B cells have recently become a focus in breast cancer pathology due to their influence on tumour regression, prognosis, and response to treatment, besides their contribution to antigen presentation, immunoglobulin production, and regulation of adaptive responses. As our understanding of diverse B cell subsets in eliciting both pro- and anti-inflammatory responses in breast cancer patients increases, it has become pertinent to address the molecular and clinical relevance of these immune cell populations within the tumour microenvironment (TME). At the primary tumour site, B cells are either found spatially dispersed or aggregated in so-called tertiary lymphoid structures (TLS). In axillary lymph nodes (LNs), B cell populations, amongst a plethora of activities, undergo germinal centre reactions to ensure humoral immunity. With the recent approval for the addition of immunotherapeutic drugs as a treatment option in the early and metastatic settings for triple-negative breast cancer (TNBC) patients, B cell populations or TLS may resemble valuable biomarkers for immunotherapy responses in certain breast cancer subgroups. New technologies such as spatially defined sequencing techniques, multiplex imaging, and digital technologies have further deciphered the diversity of B cells and the morphological structures in which they appear in the tumour and LNs. Thus, in this review, we comprehensively summarise the current knowledge of B cells in breast cancer. In addition, we provide a user-friendly single-cell RNA-sequencing platform, called “B singLe cEll rna-Seq browSer” (BLESS) platform, with a focus on the B cells in breast cancer patients to interrogate the latest publicly available single-cell RNA-sequencing data collected from diverse breast cancer studies. Finally, we explore their clinical relevance as biomarkers or molecular targets for future interventions. Full article

The incidence of left-sided colon and rectal cancer in young people are increasing worldwide, but its causes are poorly understood. It is not clear if the tumor microenvironment is dependent on age of onset, and little is known about the composition of tumor-infiltrating T cells in early-onset colorectal cancer (EOCRC). To address this, we investigated T-cell subsets and performed gene expression immune profiling in sporadic EOCRC tumors and matched average-onset colorectal cancer (AOCRC) tumors. Left-sided colon and rectal tumors from 40 cases were analyzed; 20 EOCRC (<45 years) patients were matched 1:1 to AOCRC (70–75 years) patients by gender, tumor location, and stage. Cases with germline pathogenic variants, inflammatory bowel disease or neoadjuvant-treated tumors were excluded. For T cells in tumors and stroma, a multiplex immunofluorescence assay combined with digital image analysis and machine learning algorithms was used. Immunological mediators in the tumor microenvironment were assessed by NanoString gene expression profiling of mRNA. Immunofluorescence revealed no significant difference between EOCRC and AOCRC with regard to infiltration of total T cells, conventional CD4⁺ and CD8⁺ T cells, regulatory T cells, or γδ T cells. Most T cells were located in the stroma in both EOCRC and AOCRC. Immune profiling by gene expression revealed higher expression in AOCRC of the immunoregulatory cytokine IL-10, the inhibitory NK cell receptors KIR3DL3 and KLRB1 (CD161), and IFN-a7 (IFNA7). In contrast, the interferon-induced gene IFIT2 was more highly expressed in EOCRC. However, in a global analysis of 770 tumor immunity genes, no significant differences could be detected. T-cell infiltration and expression of inflammatory mediators are similar in EOCRC and AOCRC. This may indicate that the immune response to cancer in left colon and rectum is not related to age of onset and that EOCRC is likely not driven by immune response deficiency. Full article

T cells express an actin-binding protein, drebrin, which is recruited to the contact site between the T cells and antigen-presenting cells during the formation of immunological synapses. However, little is known about the clinical implications of drebrin-expressing, tumor-infiltrating lymphocytes (TILs). To address this issue, we evaluated 34 surgical specimens of pathological stage I–IIIA squamous cell lung cancer. The immune context of primary tumors was investigated using fluorescent multiplex immunohistochemistry. The high-speed scanning of whole-slide images was performed, and the tissue localization of TILs in the tumor cell nest and surrounding stroma was automatically profiled and quantified. Drebrin-expressing T cells were characterized using drebrin⁺ T cells induced in vitro and publicly available single-cell RNA sequence (scRNA-seq) database. Survival analysis using the propensity scores revealed that a high infiltration of drebrin⁺ TILs within the tumor cell nest was independently associated with short relapse-free survival and overall survival. Drebrin⁺ T cells induced in vitro co-expressed multiple exhaustion-associated molecules. The scRNA-seq analyses confirmed that the exhausted tumor-infiltrating CD8⁺ T cells specifically expressed drebrin. Our study suggests that drebrin-expressing T cells present an exhausted phenotype and that tumor-infiltrating drebrin⁺ T cells affect clinical outcomes in patients with resectable squamous cell lung cancer. Full article

Digital quantification based on counting of individual molecules is a promising approach for different biomedical applications due to its enhanced sensitivity. Here, we present a method for the digital detection of nucleic acids (DNA and RNA) on silicon microchips based on the counting of gold nanoparticles (GNPs) in DNA duplexes by scanning electron microscopy (SEM). Biotin-labeled DNA is hybridized with capture oligonucleotide probes immobilized on the microchips. Then biotin is revealed by a streptavidin–GNP conjugate followed by the detection of GNPs. Sharp images of each nanoparticle allow the visualization of hybridization results on a single-molecule level. The technique was shown to provide highly sensitive quantification of both short oligonucleotide and long double-strand DNA sequences up to 800 bp. The lowest limit of detection of 0.04 pM was determined for short 19-mer oligonucleotide. The method’s applicability was demonstrated for the multiplex quantification of several β-lactamase genes responsible for the development of bacterial resistance against β-lactam antibiotics. Determination of nucleic acids is effective for both specific DNA in lysates and mRNA in transcripts. The method is also characterized by high selectivity for single-nucleotide polymorphism discrimination. The proposed principle of digital quantification is a perspective for studying the mechanisms of bacterial antibiotic resistance and bacterial response to drugs. Full article

Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method ‘circular fluorescent in situ hybridization’ (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples. Full article

Patients with COPD may be at an increased risk for severe illness from COVID-19 because of ACE2 upregulation, the entry receptor for SARS-CoV-2. Chronic exposure to cigarette smoke, the main risk factor for COPD, increases pulmonary ACE2. How ACE2 expression is controlled is not known but may involve HuR, an RNA binding protein that increases protein expression by stabilizing mRNA. We hypothesized that HuR would increase ACE2 protein expression. We analyzed scRNA-seq data to profile ELAVL1 expression in distinct respiratory cell populations in COVID-19 and COPD patients. HuR expression and cellular localization was evaluated in COPD lung tissue by multiplex immunohistochemistry and in human lung cells by imaging flow cytometry. The regulation of ACE2 expression was evaluated using siRNA-mediated knockdown of HuR. There is a significant positive correlation between ELAVL1 and ACE2 in COPD cells. HuR cytoplasmic localization is higher in smoker and COPD lung tissue; there were also higher levels of cleaved HuR (CP-1). HuR binds to ACE2 mRNA but knockdown of HuR does not change ACE2 protein levels in primary human lung fibroblasts (HLFs). Our work is the first to investigate the association between ACE2 and HuR. Further investigation is needed to understand the mechanistic underpinning behind the regulation of ACE2 expression. Full article