BioTechniques最新文献

Single-cell spatial technologies have emerged in recent years, enabling characterization of tissue architecture and organization at unprecedented resolution. However, computational analysis of spatial transcriptomics data remains a bottleneck for scientific discoveries in the absence of dedicated bioinformatics expertise. Here, we describe a novel cell area normalization method and workflow to annotate 15 kidney cell types from a dataset generated using NanoString's CosMx single-cell-resolution spatial transcriptomic platform. This approach enabled a comparison between two healthy kidney biopsies and two diseased samples. We validated our pipeline's accuracy using gene expression analysis, demonstrating increased sensitivity compared with other normalization methods and consistency with pathological changes observed in biopsy-proven diabetic kidney disease (DKD). Using precise cell type annotation, we observed significant changes in the proportions of podocytes and immune cells in DKD, with regional enrichment of immune cells and differential gene expression. Injured proximal tubules showed the expected upregulation of HAVCR1 and VCAM1, as well as other genes associated with diabetes, including IL18, ITGA3, and ITGB1. The workflow, now fully integrated into the BioTuring SpatialX (Lens V2.0), is available as a platform designed for users with no formal bioinformatics training, providing accessible web-based tools for spatial data analysis.

DNA barcoding of small organisms often requires significant damage or destroy specimens. To address this, the development of nondestructive DNA extraction methods that maintain specimen morphology is crucial. Here, we present a protocol using the supernatant of DESS preservation solution (20% DMSO, 250 mM EDTA, saturated NaCl), which conserve both the morphological characteristics and DNA of biological samples long-term. This method successfully conducted nondestructive barcoding of nematodes preserved in DESS and stored 10 years at room temperature. The protocol also applies to bulk environmental samples, where sediment and seagrass collected in the field are immediately preserved in DESS. This enables the subsequent isolation and individual nondestructive barcoding of meiofauna and diatoms from the preserved environmental samples in the laboratory.

The LexA-E. coli two-hybrid (LexA-E2H) system was initially developed to study interactions between microbial proteins in an Escherichia coli (E. coli) environment. We here demonstrate its utility for studying mammalian protein interactions. Specifically, this study uses LexA-E2H to provide the first direct and quantitative validation of Glucose Regulated Protein 78 (GRP78) binding to the cleaved-Prostate Apoptosis Response 4 (cl-Par-4) tumor suppressor. Furthermore, the results establish that this interaction does not require phosphorylation of either protein. MacConkey agar was used for initial detection of the interaction through colorimetric colony screening, distinguishing pale white-pink colonies (+ interaction) from red colonies (- interaction). This was followed by β-galactosidase assays for quantitative assessment. These results demonstrate the potential of the LexA-E2H system to advance human protein-protein interaction research. LexA-E2H is simple to implement, avoiding the need to culture eukaryotic cells, and bypassing interference from eukaryotic proteins. This system is ideal for laboratories with limited resources and complements conventional eukaryotic methods.

MicroRNAs (miRNAs) are considered more stable than mRNA, but the impact of progressive thawing of biological samples after freezing as may happen during shipping delays has not been quantified. To address this, we utilized digital PCR to estimate the absolute concentrations of select miRNAs following progressive thawing of human plasma and maintenance at ambient temperature. Specifically, we quantified let-7b-3p, miR-144-5p, miR-150-5p, miR-517a-3p, miR-524-5p, and miR-1283, which have varying abundance in plasma. We observed a trend indicating a decline in miRNA concentration as plasma samples were progressively thawed. Notably, miR-150-5p and miR-517a-3p were the least stable and were degraded by 32% and 52% respectively after 24 hours of ambient temperature storage. We found that the variation in sensitivity to temperature was not due to the GC content of the miRNAs nor their initial abundance, suggesting that other factors, such as protein interactors and vesicles carrying these miRNAs, may impact sensitivity.

What makes a successful core facility? While many metrics are suggested and requested to evaluate the success and impact of a core, a comprehensive understanding of the priority of the institution and core and how each metric fits into the overall strategy for the core facility is critical before determining research value or success in specific instances. A description of some different metrics commonly used to evaluate cores is presented in addition to the variable impact or interpretation of the metric in the case of different core facility structures. For example, in the case of a research - focused institution or core facility, the number and type of publications contributed to may be highly valued. Contrastingly, publications may be of less evaluative value in the case of a teaching or discovery - prioritized institution. Overall, we suggest a balanced view of core evaluation that presents each indicator in the specific context of the institution and specific core and technology.

In mitotic chromosome preparation, it is crucial to maximize chromosome yield for downstream cytogenetic analysis. Using HeLa cells as a model adherent cell, we assessed and compared the recovery of chromosomes from the entire process as well as the fraction of chromosomes that would generally become discarded in the standardly used trypsinization and mitotic-shake-off chromosome preparation methods. A higher chromosome yield for polyamine (PA) and methanol acetic acid (MAA) chromosomes was achieved using the mitotic-shake-off method compared to trypsinization. Moreover, mitotic arrest using colcemid or nocodazole gave similar PA and MAA chromosome yields in the commonly collected fractions. Interestingly, when comparing the fractions that would usually be discarded in the mitotic-shake-off, for colcemid-treated cells compared to nocodazole-treated cells, a greater number of PA chromosomes was recovered from the former. Our results show that chromosomes can be retrieved from the waste media. These recovered chromosomes display a suitable morphology in all chromosome preparations, suggesting that in conditions where high chromosome yields are required, utilizing the mitotic-shake-off method and recovering the generally discarded chromosome fraction together with the commonly used fraction would aid in maximizing chromosome yield.

Super-Resolution Radial Fluctuation (SRRF) enables live-cell super-resolution imaging, but requires careful parameter selection. Here, we quantify the impact of NanoJ-SRRF parameters on microtubule imaging using FWHM and SQUIRREL-based error mapping. Ring radius proved most critical, with values >1.0 degrading resolution and fidelity. Radiality magnification and axes in ring had minimal impact. Advanced parameters revealed pitfalls: "remove positivity constraint" degraded resolution by 43%, while gradient weighting catastrophically reduced fidelity (RSP = 0.204 ± 0.116). Temporal Radiality Average (TRA) outperformed Temporal Radiality Auto-Correlations (TRAC), milimizing artifacts. This study establishes the first evidence-based guidelines for live-cell tubulin imaging: ring radius ≤1.0, TRA mode prioritization, and avoidance of gradient weighting. Integrating FWHM and SQUIRREL offers a robust opitimization framework for cytoskeletal dynamics.

Modular cloning has transformed synthetic biology by enabling rapid assembly of standardized DNA parts, but its effectiveness is often limited to well-characterised model organisms. Here, we expand the utility of the NEBuilder^® HiFi DNA Assembly Kit (New England Biolabs) into a quasi-modular system that uses single-stranded oligonucleotide bridges to assemble reusable DNA fragments without the need for restriction enzymes. Using the anaerobic gut eukaryote Blastocystis ST7-B as a test case, we constructed and reassembled vectors with diverse promoter-terminator combinations. These results underscore the versatility of the method and its potential to accelerate genetic tool development in non-model biological systems.

Collection of the buffy coat layer from whole blood is critical for detecting rare circulating cells, such as circulating tumor cells (CTCs), which are of great diagnostic and research importance. Conventional methods for buffy coat collection often have low yields, significant erythrocyte contamination, and/or high costs limiting their utility. We developed a novel, multichannel aspiration device that provides efficient buffy coat collection with minimal erythrocyte contamination. This study employed spiked-in myeloma cells to model CTCs and evaluate device performance across a range of CTC concentrations (12, 30, and 300 cells/mL). The device demonstrated high CTC recovery rates, achieving up to 98% at high CTC concentrations and 89% at low concentrations. Immunofluorescent imaging confirmed preservation of cell morphology throughout the collection process. This convenient technology offers the potential of a low-cost alternative for buffy coat collection to be utilized in a wide range of clinical and research applications.