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PubMed2026

Post-Hoc Long-Read Sequencing Links Leukemic Mutation Status to Single-Cell Transcriptomes.

Single-cell RNA-sequencing-based characterization of cells that belong to the neoplastic clone is a major challenge in hematologic neoplasms, where malignant and normal cells coexist. Confident molecular profiling requires simultaneous analysis of gene expression and genetic mutations in individual cells, an ability that is not supported by the standard 10X Genomics workflow. Here, we systematically evaluated the potential and limitations of repurposing amplified cDNA generated during the 10X Genomics 3' workflow for post hoc genotyping of individual cells. We first established a mixed leukemic cell line system comprising one cell line with KIT point mutations and another with the BCR::ABL1 fusion gene. Targeted long-read PacBio sequencing enabled post hoc assignment of mutation data to transcriptionally profiled cells, but recovery differed between targets. Consistent with ambient RNA in microfluidics-based single-cell workflows, mutation-associated transcripts were detected in cells not expected to carry the corresponding mutations, illustrating how transcript recovery complicates cell-level genotype assignment. Target-specific thresholds mitigated this source of misclassification. In primary chronic myeloid leukemia samples, the post hoc approach detected BCR::ABL1-positive cells at diagnosis, but not during imatinib treatment. Together, we present a framework for adding mutation status to cells already profiled using the 10X Genomics workflow and highlight broader considerations for transcript-based single-cell genotyping.

BCR::ABL1↗

PubMed2024

Phenotypic targeting using magnetic nanoparticles for rapid characterization of cellular proliferation regulators.

Genome-wide CRISPR screens have provided a systematic way to identify essential genetic regulators of a phenotype of interest with single-cell resolution. However, most screens use live/dead readout of viability to identify factors of interest. Here, we describe an approach that converts cell proliferation into the degree of magnetization, enabling downstream microfluidic magnetic sorting to be performed. We performed a head-to-head comparison and verified that the magnetic workflow can identify the same hits from a traditional screen while reducing the screening period from 4 weeks to 1 week. Taking advantage of parallelization and performance, we screened multiple mesenchymal cancer cell lines for their dependency on cell proliferation. We found and validated pan- and cell-specific potential therapeutic targets. The method presented provides a nanoparticle-enabled approach means to increase the breadth of data collected in CRISPR screens, enabling the rapid discovery of drug targets for treatment.

Humans↗

PubMed2024

Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus.

Spatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with in situ reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX. We developed computational pipelines to register data from two distinct modalities. Performing both DBiT-seq and CODEX on the same tissue slide enables accurate cell typing in each spatial transcriptome spot and subsequently image-guided decomposition to generate single-cell resolved spatial transcriptome atlases. DBiTplus was applied to mouse embryos with limited protein markers but still demonstrated excellent integration for single-cell transcriptome decomposition, to normal human lymph nodes with high-plex protein profiling to yield a single-cell spatial transcriptome map, and to human lymphoma FFPE tissue to explore the mechanisms of lymphomagenesis and progression. DBiTplusCODEX is a unified workflow including integrative experimental procedure and computational innovation for spatially resolved single-cell atlasing and exploration of biological pathways cell-by-cell at genome-scale.

Journal Article↗

PubMed2026

Molecular biology and integrated strategies for activating cryptic biosynthetic gene clusters toward next-generation antibiotic discovery.

Antimicrobial resistance (AMR) has been identified as one of the 21st century's severest global public health crises. AMR led to an estimated 4.95 million deaths in 2019 and will claim 10 million lives a year by 2050 in the absence of targeted interventions. During the same period, the number of novel antibiotics discovered has decreased drastically as many researchers are rediscovering known antibiotics, non-model microorganisms are poorly understood or difficult to culture and antibiotic research and development investment has declined drastically. However, high-throughput whole genome sequencing and the subsequent application of bioinformatics in bacterial and fungal genomes have shown that a numerous of cryptic or silent biosynthetic gene clusters (BGCs) remain latent at ambient laboratory conditions since their genes are transcriptionally inactive. Cryptic BGCs represent a vast source of unique secondary metabolites, many of which may yield novel antibacterial, antifungal, anti-cancer and other potentially valuable natural products. This review discusses the biological relevance of cryptic BGCs, the major limiting factors that restricts their activation and novel strategies that have been employed to activate them and exploit their potential to produce novel natural products. The review focuses on biological approaches including CRISPR-Cas mediation for the activation of cryptic BGCs, promoter engineering, pathway refactoring, and heterologous expression; biochemical strategies such as Osman, OsMAC, Precursor Feeding, Chemical Elicitation, Epigenetic Regulation and Co-cultivation and technology-based strategies such as Genome mining, Microfluidic Cultivation systems, High-Throughput Screening, Metabolomics, Molecular Networking and Artificial Intelligence and Machine Learning based prediction of BGCs and their metabolites. The use of multi-omics technologies combined with synthetic biology to achieve better discovery, characterization and large-scale production of novel natural products is also discussed herein. Finally, we will talk about the ecological significance and evolutionary advantage of cryptic BGCs' role in interactions between microorganisms, such as competition, communication, symbiosis and environmental adaptability, so as to provide a useful background for accelerating next-generation antibiotics.

CRISPR-Cas activation↗

PubMed2026

Induced pluripotent stem cell reprogramming: methodological evolution and challenges in clinical translation.

Cell reprogramming can transform somatic cells into induced pluripotent stem cells providing a platform for patient-specific disease modeling, drug screening and regenerative medicine research. Since the advent of OKSM-mediated reprogramming, the system of technical approaches has evolved continuously - from integrated viral vectors to non-integrated episomal systems and, more recently, chemical reprogramming and CRISPR approaches. The simultaneous advances in single-cell multi-omics, biomaterials engineering, and artificial intelligence have further refined the controllability and precision of the reprogramming process. Despite these innovations, problems persist that hinder clinical translation: incomplete epigenetic resetting, ongoing clonal heterogeneity, genomic instability in long-term culture, and the lack of standardized Good Manufacturing Practice protocols for large-scale manufacturing. This review summarizes the trajectory of iPSC reprogramming technologies, with special emphasis on the translational applicability of each modality. We evaluated viral and nonviral delivery systems, chemical reprogramming, strategies that aid gene editing, and emerging engineering platforms, including microfluidics, smart biomaterials, and artificial-intelligence-driven process optimization. We further identify the core "translational triltrilas", namely, the inherent tradeoffs between security, homogeneity, and scalability, and propose a comprehensive strategy to overcome these bottlenecks. By linking basic mechanistic understandings with industrial and regulatory considerations, this review aims to provide a route for transitioning iPSC technology from a laboratory tool to a clinically viable manufacturing platform.

clinical translation↗

PubMed2024

Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality.

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus-host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

Nipah Virus↗

PubMed2026

Bridging Organ-on-a-Chip and Omics: A Multi-Dimensional Frontier in Biomedical Research.

Organ-on-a-Chip (OOC) technology offers a powerful platform for replicating human tissue-specific microenvironments, thereby narrowing the translational gap between conventional biomedical models and actual human physiology. Concurrently, omics technologies deliver comprehensive molecular-level insights into biological systems. This review highlights the transformative potential of integrating OOC platforms with high-throughput omics methodologies. We systematically examine the classification, structural configurations, and engineering principles underlying OOC systems, alongside the defining attributes of key omics domains-genomics, transcriptomics, proteomics, and metabolomics. The convergence of dynamic OOC models with advanced omics technologies enables high-resolution, multi-dimensional analyses across numerous biomedical applications, including drug metabolism, disease mechanisms, environmental toxicity assessments, and host-microbiome interactions. This interdisciplinary integration is driving a paradigm shift in precision and translational medicine. However, several challenges remain to be addressed, such as the development of whole-organ mimetics, adaptation of sample collection techniques, and real-time artificial intelligence-based integration of biosensor data with multi-omics datasets. Addressing these hurdles will be vital for unlocking the full potential of this technological synergy in biomedical science.

Multiomics↗

PubMed2026

Liquid Biopsy Differentiation of Pancreatic Cancer From Non-Cancerous Pancreatic Disease Using Dielectrophoresis-Recovered Nanoparticles Carrying Cell-Free DNA and Protein Biomarkers.

Cancer-derived extracellular vesicle (EV) nanoparticles carry important biomarkers but are difficult to recover from plasma, making EV-based diagnostics a challenge for clinical settings. Here, we demonstrate nanoparticle-based detection of pancreatic cancer using dielectrophoresis (DEP) nanoparticle recovery technology, which purifies nanoparticles from undiluted plasma and quantifies associated biomarkers. We combined both nanoparticle recovery and biomarker quantification on a single device by simultaneously collecting cell-free DNA nanoparticles and EVs followed by on-chip biomarker fluorescent staining for DNA and Glypican-1. Using a blinded cohort, these biomarkers differentiated pancreatic cancer from benign pancreatic diseases, including cysts, pancreatitis, and precancerous low-grade intraductal papillary mucinous neoplasm (IPMN) lesions, with a sensitivity of 0.92, a specificity of 0.83, and an AUC of 0.93. The AUC increased to 0.97 for patients over 50 years old. This is higher than the standard invasive endoscopic ultrasound-guided fine needle aspiration tissue biopsy procedure (AUC 0.79). This study is among the first demonstrating a combined threshold of DNA and protein levels that can distinguish pancreatic cancer from its precursor IPMN lesions. We also demonstrated the detection of early-stage pancreatic cancer and high-grade in situ precancerous lesions. This DEP-based technique shows that multiple types of cancer-derived nanoparticles can be quickly and easily recovered from plasma making it promising for future clinical diagnostics.

Humans↗

PubMed2025

Mismatch-introduced crRNA guided PCR-CRISPR/Cas12a platform improves EGFR point mutation detection in single tumor cell.

Dynamic monitoring of epidermal growth factor receptor (EGFR) mutations is essential for the early identification of resistance and treatment adaptation. Single-cell heterogeneity analysis is crucial for precision cancer medicine, yet sensitive and specific detection methods for individual tumor cells remain challenging. Here, we develop a PCR-CRISPR/Cas12a platform enhanced by the incorporation of mismatched base in crRNA at specific site for single-cell point mutation detection. This platform demonstrated high specificity and sensitivity, detecting point mutation at a frequency of 0.1% and in as low as 1.02 ng of genomic DNA, which represents an improvement over the amplification-refractory mutation system PCR (ARMS-PCR). Notably, the accuracy of the platform is highly consistent with next-generation sequencing (NGS), as evidenced by Kappa test values surpassing 0.9. By utilizing a conical-pore membrane with optimized porosity for single circulating tumor cell (CTC) enrichment, our platform enables point mutations detection in individual tumor cells, offering potential enhancements in precision and reliability for EGFR mutation analysis. This novel methodology holds potential for more accurate and personalized cancer treatment strategies.

Humans↗

PubMed2026

Recent advancements in exosomal content analysis: the future of liquid biopsy.

Exosomes are widely acknowledged as an essential agent that carries biomarkers for specific diseases, representing the molecular status of their parent cells and providing extremely useful diagnostic insights. They can be isolated from different body fluids and contain a range of cargo molecules, including proteins, lipids, metabolites, and nucleic acids. Recent advancements in technology have greatly accelerated exosome research. Proteomics provides protein signatures linked to many pathological conditions, enabling quick and clinically scalable diagnostic tools, whereas high-throughput RNA-sequencing can be used to perform detailed transcriptome profiling. Exosomal biomarkers are showing promising clinical results in early detection of neurological diseases, infectious and cardiovascular disorders, oncology, and other medical conditions, hence accelerating therapeutic monitoring. Despite these advances, several challenges continue to hinder clinical translation including the lack of standardized isolation protocol, variability in exosome yield and purity, biological heterogeneity, and limited large-scale clinical validation. Addressing these limitations will be critical for the successful integration of exosome-based liquid biopsy into routine clinical practice. Overall, exosomes having significant potential as diagnostic tool, represent a transformative horizon in biomedical liquid biopsy research to redefine the landscape of less-invasive diagnostics and tailored clinical applications.

Humans↗

PubMed2026

First insights into the role of evolutionary history in shaping venom composition of Vipera ammodytes.

Understanding intraspecific venom variation requires distinguishing the contributions of neutral population history from natural selection. This study aims to determine whether venom variation in Vipera ammodytes species complex is structured across eight phylogenetic groups. Despite a complex evolutionary history, venom composition did not differ among phylogenetic groups within the analytical framework used, suggesting that shared ancestry alone does not explain venom variation. Whether local adaptation to environmental conditions explains the observed variation remains an open question for future studies.

Animals↗

PubMed2026

Advances in Single-Molecule Immunoassay: From Counting Strategies to CRISPR-Enhanced Biosensing.

Single-molecule immunoassays (SMIs) overcome the sensitivity limitations of conventional bulk measurements by enabling a paradigm shift from analog to digital signal readouts, thereby facilitating highly sensitive quantification of ultra-low-abundance biomarkers for precision diagnostics. This review provides a systematic overview of recent advances in SMI technologies and the conceptual framework underlying their evolution. First, discretization strategies for single-molecule counting are classified into hard discretization, based on physical confinement, and soft discretization, based on spatiotemporal isolation, within heterogeneous and homogeneous assay systems, respectively. The fundamental mechanisms by which these strategies mitigate diffusion limitations and enhance signal-to-noise ratios are discussed. Second, the integration of SMIs with CRISPR-based diagnostic systems (CRISPR-dx) is examined, with particular emphasis on their complementary roles in target recognition and signal amplification. Finally, recent applications of SMIs in the diagnosis of oncological, neurological, infectious, and cardiovascular diseases are summarized, along with a critical discussion of current engineering challenges and future directions toward clinical translation.

Immunoassay↗