Cognitive aging is a highly heterogeneous process, with some individuals preserving stable cognitive performance across the lifespan while others exhibiting pronounced decline. This marked interindividual variability indicates that chronological age alone is a poor predictor of cognitive health. Rather than reflecting uniform degeneration, cognitive aging emerges from divergent biological trajectories spanning molecular, cellular, and network levels. In this review, we synthesize emerging biological hallmarks of healthy cognitive aging, emphasizing studies that characterize longitudinal cognitive trajectories in humans or distinguish aged individuals who retain learning capacity from those who do not. We focus on the medial temporal lobe, a region critical for episodic memory and spatial navigation, and examine how variability in its integrity contributes to distinct cognitive outcomes. Across species, convergent evidence suggests that cognitive decline is more closely linked to alterations in network regulation and synaptic plasticity than to overt neuronal loss. We identify key mechanisms shaping individual trajectories, including large-scale network organization, excitation–inhibition balance, neuromodulatory tone, glial and vascular regulation, adult hippocampal neurogenesis, and cellular homeostasis. These processes operate within an interconnected system in which disruptions in core regulatory mechanisms can propagate across levels of organization. Together, this synthesis supports a system-level framework in which cognitive resilience depends on the preservation of coordinated network dynamics. We advocate for longitudinal, multidimensional approaches to identify early shifts in regulatory balance and inform strategies to maintain cognitive function across the lifespan.

Imaging-based spatially resolved transcriptomics can localize transcripts within tissue sections in three dimensions. However, cell segmentation, which assigns transcripts to cells, is usually performed in two dimensions and spatial doublets in the vertical dimension result in segmented cells containing transcripts originating from multiple cell types. Here we present a computational tool called ovrlpy that identifies overlapping cells, tissue folds and inaccurate cell segmentation by analyzing transcript localization in three dimensions.

Rolling circle amplification (RCA) combined with padlock probes presents a promising tool for direct detection and genotyping of viral RNA, offering advantages over conventional methods like RT-PCR. This isothermal process enables highly sensitive and specific amplification of nucleic acids without the need for thermal cycling, making it suitable for point-of-care applications. In this study, we demonstrate a microfluidic and RCA-based method for the direct detection of SARS-CoV-2 RNA and variant profiling, bypassing the reverse transcription step. Our approach allows for the identification of single nucleotide polymorphisms (SNPs) specific to viral variants, enhancing the detection sensitivity through the circle-to-circle amplification (C2CA) technique. This methodology shows potential as a robust, cost-effective platform for viral diagnostics, capable of being fully automated and integrated with miniaturized detection systems for efficient use in both resource-rich and resource-limited settings.

Background T cell receptor (TCR) binding properties have been related to a wide range of pathological conditions, including infections, autoimmunity and cancer. Characterising the TCR repertoire is of great biomedical interest but it has been challenging due to its high structural diversity.

Methods In situ sequencing (ISS) is a suitable technique for spatial cell typing and linking gene patterns directly to specific histopathological features of large biopsy areas. We applied ISS through the commercial Xenium platform, with the addition of a custom panel specifically designed for TCR gene detection. Based on the IMGT database, we selected unique target sequences for TCR genes encoding the constant, variable and joining TCR chains. Additionally, we developed an analysis pipeline for the assignment of putative clonotypes based on simultaneous expression of alpha and beta variable TCR chains (TCRVβ/Vα pairs) at the single-cell level.

Findings Our approach captured specific immune cell distributions in relation to the individual sample clonality, as well as regional dominance of certain TCRVβ/Vα pairs in surgical non-small cell lung cancer (NSCLC) specimens and matching lymph node samples. Furthermore, we were able to study the spatiotemporal evolution of TCR repertoire on longitudinal FFPE biopsies from patients with breast cancer, during neoadjuvant treatment.

Interpretation This study highlights the implementation of target-based spatially resolved transcriptomics for the spatial characterisation of TCRVβ/Vα pairs at the single-cell level, without the need for prior sequencing. Our approach allows for spatial immune characterisation of diagnostic tissue samples with emphasis on T cell biology and accompanying T cell diversity.

Background: Pathogen detection in primary care is crucial not only to identify viruses like SARS-CoV-2 but also for antibiotic-resistant bacteria. While microfluidic devices enable point-of-care diagnostics, they often lack sufficient sensitivity. On-chip isothermal amplification techniques, such as padlock probing-based rolling circle amplification (PLP-RCA), can enhance specificity and sensitivity while keeping device complexity low. However, integrating PLP-RCA on-chip requires precise optimization of enzyme concentrations, flow conditions, and target capture to achieve its full potential. Results: This study demonstrates a microfluidic RCA assay using porous agarose microbeads as a solid-phase capture, packed inside a microfluidic device. Various target capture strategies were systematically compared and quantitatively investigated, progressing from single-stranded synthetic DNA oligonucleotides to double-stranded Staphylococcus aureus genomic DNA. The best strategy for double-stranded Staphylococcus aureus genomic DNA used a primer bound to the beads that capture the PLP and the target genomic DNA. The system integrates an amorphous-hydrogenated silicon (a-Si:H) thin film p-i-n photodiode and a high-pass interference filter, enabling on-chip fluorescence signal acquisition of amplicons. This integration allows for a fully functional PLP-RCA assay on-chip, along with the added merits of device portability and compatibility with clinical demands. Significance and novelty: This study systematically evaluates single- and double-stranded target capture for on-chip PLP-RCA assays. It demonstrates the successful integration of microfluidics with a solid-phase capture medium and fluorescence detection system. The findings highlight the potential of this platform for developing sensitive, portable pathogen detection devices suited for clinical applications.”

Background.  Glioblastoma (GB), particularly IDH-wild type, is the most aggressive brain malignancy with a dismal prognosis. Despite advances in molecular profiling, the complexity of its tumor microenvironment and spatial organization remains poorly understood. This study aimed to create a comprehensive single-cell and spatial atlas of GB to unravel its cellular heterogeneity, spatial architecture, and clinical relevance.

Methods.  We integrated single-cell RNA sequencing data from 26 datasets, encompassing over 1.1 million cells from 240 patients, to construct GBmap, a harmonized single-cell atlas. High-resolution spatial transcriptomics was employed to map the spatial organization of GB tissues. We developed the Tumor Structure Score (TSS) to quantify tumor organization and correlated it with patient outcomes.

Results.  We showcase the applications of GBmap for reference mapping, transfer learning, and biological discoveries. GBmap revealed extensive cellular heterogeneity, identifying rare populations such as tumor-associated neutrophils and homeostatic microglia. Spatial analysis uncovered 7 distinct tumor niches, with hypoxia-dependent niches strongly associated with poor prognosis. The TSS demonstrated that highly organized tumors, characterized by well-defined vasculature and hypoxic niches, correlated with worse survival outcomes.

Conclusions.  This study provides a comprehensive resource for understanding glioblastoma heterogeneity and spatial organization. GBmap and the TSS provide an integrative view of tumor architecture in GB, highlighting hypoxia-driven niches that may represent avenues for further investigation. Our resource can facilitate exploratory analyses and hypothesis generation to better understand disease progression.

Recent advancements in spatial transcriptomics have been largely triggered by two high-resolution technologies: Visium-HD and Xenium in-situ. While sequencing-based Visium HD features a refined bin size of 2 µm and transcriptome wide coverage, Xenium in-situ is a targeted imaging-based detection technology with sub-micron resolution. Herein we use a publicly available lung dataset which contains Visium-HD and Xenium-5K data generated on identical tissue slides to make a bona-fide technical comparison aligned with thorough pathological annotations. Whilst Visium-HD offers a broader gene coverage for detection and likely detects more tumor subclones, Xenium-5K achieves comparable results when robust clustering algorithms are applied. Importantly, from the pathological point of view, the single-cell segmentation accuracy is essential when analyzing irregularly shaped cells, where Xenium may be in favor. At the opposite side, although Xenium-5K based cell segmentation to delineate immune cells, normal lung, and vasculature at cell resolution is decent, it relies on fluorescent signals for transcript detection, which is challenging in heavily pigmented tissues such as melanoma or dust-laden alveolar macrophages, an application scenario for which Visium HD may stand out. From this perspective, pathological derived factors are the prior consideration for selecting an appropriate ST approach under difference research settings including cancer.

The amniote pallium contains sensory circuits that are structurally and functionally equivalent, yet their evolutionary relationship remains unresolved. We used birthdating analysis, single-cell RNA and spatial transcriptomics, and mathematical modeling to compare the development and evolution of known pallial circuits across birds (chick), lizards (gecko), and mammals (mouse). We reveal that neurons within these circuits’ stations are generated at varying developmental times and brain regions across species and found an early developmental divergence in the transcriptomic progression of glutamatergic neurons. Our research highlights developmental distinctions and functional similarities in the sensory circuit between birds and mammals, suggesting the convergence of high-order sensory processing across amniote lineages.

Oligodendroglia are the responsible cells for myelination in the central nervous system and their involvement in Parkinson’s disease (PD) is poorly understood. We performed sn-RNA-seq and image-based spatial transcriptomics of human caudate nucleus and putamen (dorsal striatum) from PD and control brain donors to elucidate the diversity of oligodendroglia and how they are affected by the disease. We profiled a total of ~ 200.000 oligodendroglial nuclei, defining 15 subclasses, from precursor to mature cells, 4 of which are disease-associated. These PD-specific populations are characterized by the overexpression of heat shock proteins, as well as distinct expression signatures related to immune responses, myelination alterations, and disrupted cell signaling pathways. We have also identified impairments in cell communication and oligodendrocyte development, evidenced by changes in neurotransmitter receptors expression and cell adhesion molecules. In addition, we observed significant disruptions in oligodendrocyte development, with aberrant differentiation trajectories and shifts in cell proportions, particularly in the transition from mature oligodendrocytes to disease-associated states. Quantitative immunohistochemical analysis revealed decreased myelin levels in the PD striatum, which correlated with transcriptomic alterations. Furthermore, spatial transcriptomics mapping revealed the distinct localization of disease-associated populations within the striatum, with evidence of impaired myelin integrity. Thus, we uncover oligodendroglia as a critical cell type in PD and a potential new therapeutic target for myelin-based interventions.

The Xenium In Situ platform is a new spatial transcriptomics product commercialized by 10x Genomics, capable of mapping hundreds of genes in situ at subcellular resolution. Given the multitude of commercially available spatial transcriptomics technologies, recommendations in choice of platform and analysis guidelines are increasingly important. Herein, we explore 25 Xenium datasets generated from multiple tissues and species, comparing scalability, resolution, data quality, capacities and limitations with eight other spatially resolved transcriptomics technologies and commercial platforms. In addition, we benchmark the performance of multiple open-source computational tools, when applied to Xenium datasets, in tasks including preprocessing, cell segmentation, selection of spatially variable features and domain identification. This study serves as an independent analysis of the performance of Xenium, and provides best practices and recommendations for analysis of such datasets.