Cell Reports Methods最新文献

Antimicrobial resistance (AMR) poses a significant threat to public health. Rapid and accurate antimicrobial sensitivity testing is essential to guide effective treatment. Here, we present "simplified 5PSeq" (s5PSeq), a streamlined protocol for profiling 5' monophosphorylated (5'P) mRNA degradation intermediates that reflect ribosome dynamics in vivo. By capturing antibiotic-induced, context-specific ribosome stalling events, s5PSeq provides a molecular proxy for bacterial growth inhibition-offering a molecular phenotypic readout without the need for culturing. s5PSeq reduces library preparation time to under 4 h and incorporates a novel rRNA blocking strategy. We demonstrated its clinical utility by identifying erythromycin-resistant and sensitive Clostridioides difficile clinical isolates. Combining s5PSeq with real-time nanopore sequencing enables fast AMR diagnosis with as few as 3,000 reads. In addition to simplifying the study of 5'P co-translational mRNA decay, our work suggests that utilizing information-rich phenotypic molecular readouts can significantly improve AMR diagnostics.

Clustering is commonly used in single-cell RNA sequencing (scRNA-seq) to assess cellular heterogeneity, but standard methods often require user-specified heuristics and rely on post-selective differential expression analyses, which often lead to inflated false discovery rates. Here, we present NCLUSION: a nonparametric infinite mixture model that leverages Bayesian sparse priors to identify marker genes and cluster single-cell expression data simultaneously. NCLUSION uses a variational inference algorithm, which enables it to scale up to millions of cells. Through simulations and analyses of publicly available scRNA-seq studies, we demonstrate that NCLUSION (1) matches the performance of other state-of-the-art clustering techniques with significantly reduced runtime and (2) provides statistically robust and biologically relevant transcriptomic signatures for each of the clusters it identifies. Overall, NCLUSION represents a reliable hypothesis-generating tool for understanding patterns of expression variation present in single-cell populations.

Lipid vesicles are important as minimal model systems for cellular compartmentalization. They drive major advances in deciphering biological mechanisms by molecular reconstitution; provide rational solutions for primitive compartmentalization in origin-of-life studies; form the basis of synthetic cells and drug delivery vehicles. The emulsion method is a well-established route for producing bilayer lipid vesicles. However, the application of this method in microfluidics requires complex and specialized machinery. The bulk method suffers from the need to physically manipulate the vesicles through oil layers for characterization that can damage the vesicles. Given this, we present a facile and robust method for on-chip production and manipulation of lipid vesicles by the emulsion method. We prepared a simple device that allows preparation, imaging, and collection of activated lipid vesicles. This technique combines minimal processing steps with maximum flexibility in lipid vesicle production and manipulation with direct imaging, thus fast-tracking production lines across disciplines.

Vocal fold epithelial cells (VFEs) serve critical physiologic and immunologic functions at the boundary between the upper and lower airways but are difficult to maintain and expand in primary cultures. This technical challenge has impeded progress in VFE biology as well as cell banking for translational applications. Here, using primary human VFEs, we show that simultaneous inhibition of transforming growth factor β (TGF-β), Rho-associated protein kinase (ROCK), and Notch signaling with a small-molecule inhibitor cocktail enables rapid proliferation, successful passaging, and long-term expansion while preserving the core epithelial phenotype. Under anchorage-independent culture conditions, VFE progenitors generate clonal spheres that can be expanded over multiple generations; sphere-dissociated VFEs then revert toward their original phenotype, which includes the ability to form stratified squamous epithelium in organotypic cocultures. Both pathway-inhibited and sphere-cultured VFEs exhibit mechanistically appropriate remodeling of the cellular proteome. These advances offer a robust toolkit for upper airway mucosal biology and regenerative medicine.

Wide-field (WF) neurons of the tectopulvinar pathway integrate retinal and cortical inputs via large dendritic arbors crucial for rapid visual motion detection. Previous studies identified potential marker genes for mouse WF neurons. Here, we validate CBLN2 as a molecular marker of the tree shrew WF neurons and construct AAVs that exploit CBLN2 promoter to selectively target WF neurons across species. Using intersectional genetics in the tree shrew, we show that WF neuron dendrites receive a distinct pattern of VGluT1+ and VGluT2+ inputs based on their distance from the cell body in the dorsoventral axis of the superior colliculus (SC). This represents the first example of a viral tool derived from the tree shrew genome for cell-type-specific targeting across species. Our results provide a foundation for studying SC circuitry in higher-order mammals and for extending this approach to additional conserved cell types in the SC and other brain regions.

Understanding the structure-function relationships across neurons is challenging, particularly when circuits are composed of dozens of distinct cell types. We refined an approach, called "projection targeting with phototagging", that allows simultaneous elucidation of the projections, morphology, and visual response properties of diverse retinal ganglion cell (RGC) types in the mammalian retina. The approach combines retrograde virally mediated phototagging of RGCs, microscopy, and large-scale multi-electrode array (MEA) measurements. Importantly, the approach does not rely on transgenic animals and thus is potentially generalizable across species. We validated this approach in rats by targeting retinal projections to the superior colliculus (SC). We showed that multiple RGC types project to the SC and that these results in rats align well with prior findings from transgenic mouse studies.

CRISPR-Cas9 is rapidly expanding across diverse organisms. Among these advances, in-frame knockins of reporter genes have become essential for studying gene expression and protein localization. However, in hemimetabolan insects such as the German cockroach Blattella germanica, a phylogenetically basal and relevant pest species, functional fusion proteins have remained technically difficult to obtain. We present a streamlined gene-editing strategy to knock in a reporter gene in-frame with the distal-less gene, generating a functional fusion protein in B. germanica. By combining direct parental CRISPR with donor constructs designed for homology-directed repair carrying the mCherry gene, we successfully achieved targeted integration at the distal-less locus. The resulting fusion protein was functional and heritable and enabled live visualization of Distal-less protein distribution, showing fluorescence in developing appendages and the nervous system. This simple and robust methodology opens the door to generating fusion proteins in non-model insects, providing a valuable molecular tool for ecological, developmental, and pest-management research.

Cells continuously communicate through dynamic cell-cell contacts. Tools for visualizing these dynamic interactions in living cells are essential to the study of fundamental biological processes in multicellular organisms. Here, we present two fluorescent indicators, Gachapin and Gachapin-C, for visualizing dynamic cell-cell contact. Gachapin visualizes not only static but also dynamic contacts. Multiplexed imaging combining green Gachapin with spectrally distinct indicators allows simultaneous monitoring of contact dynamics, cytoskeletal assembly, and intracellular signaling during cell movement. Furthermore, the formation and disruption of contacts between neuronal processes can be visualized. Gachapin-C enables contact visualization with a single indicator component, whereas previous indicators required two components introduced into different cells. This feature allows Gachapin-C to monitor contacts between processes originating from a single cell. We expect Gachapin and Gachapin-C will serve as useful tools for providing deeper insights into cell-cell contact-mediated processes.

Many non-model rodent species are inaccessible to genetic engineering due to our limited understanding of their reproductive biology. Here, we present a low-cost, camera-based estrous-tracking technology that enables transgenesis in the white-footed mouse Peromyscus leucopus, a key reservoir for Lyme disease. We demonstrate the efficient generation of pregnant and pseudopregnant mice via timed ovulation, provide protocols for embryo generation, cultivation, microinjection, and transplantation as well as an accurate developmental timeline, and report the first engineered Peromyscus. The same technology successfully tracked conserved estrous-linked cycling behavior in other rodents, including hamsters. Finally, estrous tracking differentiated reproductively healthy, geriatric female Peromyscus from those with declining fertility based solely on their activity, providing a non-invasive method for studying reproductive senescence. Collectively, these tools represent a critical resource for engineering non-model rodents, advance the long-lived Peromyscus as a model organism, and will prove essential to heritably immunizing wild rodent populations against Lyme disease.

All-optical strategies enable identification of functional neuronal ensembles with calcium imaging and replay/alter their spatiotemporal activity with optogenetics to decipher their behavioral implications. We previously developed a fiber-coupled microscope enabling two-photon (2P) functional imaging and 2P holographic photostimulation with near-single-cell resolution in freely moving mice: 2P-FENDO. Here, we present a significantly optimized 2P-FENDO-II system that achieves a four-times-larger field of view and a more homogeneous light distribution across the field of view, both for imaging and photostimulation, while achieving better flexibility and thus optimal adaptation to the study of freely moving mice. We demonstrate the performance and versatility of 2P-FENDO-II in experiments targeting the somatosensory cortex, the visual cortex, or the cerebellar cortex, in which we show concomitant calcium imaging with jGCaMP7s and optogenetic control with ChRmine. These enhancements establish 2P-FENDO-II as a groundbreaking tool for all-optical interrogation of neuronal circuits on large volume in naturalistic situations.