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 and as well as other genes associated with diabetes, including , , and . 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.

RNA sequencing (RNA-Seq) is an essential assay for studying transcriptome profiling. Ribosomal RNA (rRNA) comprises more than 80-90% of total cellular RNA; efficient removal is essential for accurately capturing transcriptomes, particularly to sequence low-abundance mRNAs. Inefficient rRNA removal during library preparation can result from variations in sample quality, preparation methods, and handling. Estimating rRNA content in RNA-Seq libraries pre-sequencing is therefore challenging due to the absence of a reliable and cost-effective assessment method. This study addresses the issue by introducing a scalable real-time quantitative polymerase chain reaction (qPCR) based assay targeting human 18S rRNA to evaluate rRNA depletion efficiency of RNA-Seq libraries pre-sequencing. qPCR efficiency was optimized using serial dilutions of Universal Human Reference (UHR) control, and Ct thresholds were established using pilot data from 644 libraries. Following this optimization, analysis of 1748 human Total RNA-Seq libraries and 445 Poly A + two widely used RNA-Seq library methods, demonstrated a strong correlation between 18S rRNA qPCR results and post-sequencing rRNA rates. This assay was also used to evaluate the performance of Oligo (dT) beads from four different vendors to enrich mRNA. This 18S rRNA qPCR assay is a cost-effective, scalable approach for reliably predicting rRNA read percentage in RNA-Seq libraries pre-sequencing.

Despite increasing rates of metabolic syndrome, cancer treatment regimens often ignore the underlying metabolic dysfunction in patients. A high proportion of cancer patients have metabolic dysfunction, and caloric restriction has shown the potential to improve cancer treatment response. However, preclinical efficacy is hindered by inconsistent protocols. We demonstrated that a CR (caloric restriction) diet enhances radiotherapy outcome by promoting apoptosis and downregulating cell survival pathways in mice. However, CR animal models implementation greatly differs in previous cancer studies, hindering its clinical translation. Here, we propose an effective CR protocol that safely achieves a 30% caloric reduction in two weeks. Week one involves single housing the mice and taking individual caloric intake measurements across four days. Week two involves weaning the mice to a 30% reduction by reducing their caloric intake by 10% every other day, which allows for a safe reduction. This protocol integrates individualized intake measurement, gradual weaning, and real-time monitoring-features rarely combined in prior models-offering a reproducible and translationally relevant framework for cancer studies.

This study examines the stability of human mRNA and DNA in stool samples for noninvasive gastrointestinal disease detection. While stool samples are valuable for diagnosing conditions like inflammatory disorders and colorectal cancer, mRNA instability poses significant challenges, risking false-negative results. To investigate this, 97 stool samples were treated with a specialized stabilizing solution and stored at room temperature, with analyses conducted on Day 1 and Day 15. The research aimed to improve storage protocols for enhanced reliability in mRNA diagnostics, aiding in personalized medicine and biomarker discovery. Results showed variability in total nucleic acid yields, increasing from Day 1 (mean 112 ng/µL) to Day 15 (mean 165 ng/µL), highlighting the benefits of improved homogenization and bacterial lysis. Human DNA remained stable over the 14-day period. For RNA stability, three mRNA markers were analyzed: Carcinoembryonic Antigen (CEACAM5), Prostaglandin-Endoperoxide Synthase 2 (PTGS2) and cortactin (CTTN). Both CEACAM5 (p=0.064) and PTGS2 (p=0.79) maintained stability, while CTTN showed a statistically significant but only modest reduction in expression (p < 0.0001). Overall, the stabilization buffer proved effectiveness in preserving nucleic acids and provided insights into mRNA marker stability over time.

Three-dimensional (3D) cell culture is a more physiologically relevant model for drug development than two-dimensional (2D) cell culture. A common method to culture cells in 3D consists in embedding cells in synthetic or animal-based matrices that provide structural support for cell growth. They partially mimic conditions and enable scalable culture. Here, we introduce an alcohol-based olution for arvesting rganoids fficiently, denoted . We tested its harvesting potential on 2 cell lines grown as spheroids and 2 patient-derived organoids. It enables rapid, high-yield cell recovery, at room temperature (RT), and bypasses prolonged cold incubation of standard protocols. It preserves 3D structure and growth in subsequent passages.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used for nucleic acid quantification. The use of technical triplicates in RT-qPCR aims to minimize variability and improve reliability but increases reagent consumption, labor, and time. This study systematically evaluates the necessity of technical replicates by analyzing 71,142 cycle threshold (Ct) values from 1,113 RT-qPCR runs across three instruments, two detection chemistries, and 30 operators. Variability within replicates was assessed using metrics such as the coefficient of variation (CV), while the impacts of operator expertise, detection chemistry, instrument calibration, and initial template concentration were explored. The findings challenge the assumption that variability increases at low template concentrations, revealing no correlation between Ct values and CV. While inexperienced operators exhibited slightly higher variability, their replicates were still consistent, with acceptable CVs and low outlier frequencies. Dye-based detection showed greater variability than probe-based. Time since calibration had negligible effects on replicate consistency. Notably, duplicate or single replicates sufficiently approximated triplicate means. These results challenge traditional assumptions about RT-qPCR variability and provide a data-driven framework for optimizing experimental design. This study offers potential for resource savings without compromising data quality, particularly in high-throughput applications or laboratories with limited funds. The data underlying this article are available at https://doi.org/10.5281/zenodo.15072870.

Protein phosphatase 2A (PP2A), a pivotal serine/threonine phosphatase, plays a crucial role in cellular regulation and tumor suppression. Dysregulation of PP2A complex, particularly the Aα subunit and B56 family, is linked to malignancies through altered substrate interactions, exemplified by c-MYC dynamics. Given the challenges in identifying PP2A substrates-owing to the enzyme's expansive substrate range, transient interaction profiles, and complex regulatory mechanisms-we employed bioluminescence resonance energy transfer (BRET) sensors. These advanced molecular tools facilitate the real-time detection of protein-protein interactions within live cells. This investigation details the creation and application of a novel PPP2R5A (B56α) BRET sensor tailored for cytosolic and nuclear environments, effectively distinguishing specific PP2A interactions. The nuclear sensor, enhanced with a nuclear localization signal, enabled probing of targets like c-MYC. The dual compartmental utility of these sensors underscores their significant potential in elucidating PP2A's regulatory roles and their implications in oncogenesis. Our study highlights the efficacy of BRET sensors in formulating precision therapeutic strategies. This advancement provides a robust framework for deeper investigations into the multifaceted roles of PP2A in both normal physiological and pathological contexts, paving the way for future explorations into its intricate molecular interactions.

Mitophagy, a crucial mitochondrial quality control system for cellular stress adaptation, is a key focus in pathophysiology and drug discovery. Developing a simple and versatile mitophagy flux assay is vital for advancing our understanding of cellular responses. Addressing a gap in systematic methods, we employ the photoactivatable fluorescent protein mito-Kaede in C2C12 myocytes, demonstrating its remarkable versatility in quantifying mitophagy flux responses under various stimuli, including carbonyl cyanide m-chlorophenyl hydrazone (CCCP), TNF-α, lipopolysaccharide (LPS), and hypoxia. This study underscores the validity and distinctive advantages of the mito-Kaede assay through comparative analysis with conventional assays including Western blotting (WB), potentially providing valuable insights for both mitophagy flux analysis and drug development.

Total protein isolation followed by quantitation using a colorimetric method, such as the bicinchoninic acid (BCA) assay is a common laboratory protocol. Protein concentrations are determined by comparing extracted samples to a standard curve generated from serial dilutions of a reference protein, such as bovine serum albumin (BSA). This study aimed to identify the most reproducible and accurate method for quantifying protein concentrations in an experimental series over time. We analyzed the effect of serial freeze-thaws, inter-person and intra-person variability in standard preparation and assay execution. Absorbance was measured at 565 nanometers (nm) using an Epoch Microplate Spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA) with Gen5 Data Analysis software. The most consistent and accurate method for determining the protein concentrations over time is to prepare a large batch of diluted BSA standards, aliquot them into small portions, and store them frozen.

Circulating cell-free DNA (ccfDNA) can be found in blood and other biofluids and is a minimally invasive biomarker for several pathological processes. As tumors become more invasive, an increasing amount of circulating tumor DNA (ctDNA) is also shed into the peripheral circulation. Combined analysis of ccfDNA and ctDNA has demonstrated prognostic and predictive value in metastatic disease. However, localized tumors shed significantly less ccfDNA/ctDNA and accurate detection remains a technical challenge. To overcome this barrier, droplet preamplification has been used to perform robust multiplexed analysis of low-input samples. To reduce false positives, it is essential to use a high-fidelity polymerase with 3'-5' exonuclease activity. However, attempts to combine high-fidelity polymerases with commercial droplet digital chemistries have had limited success. There is also no standardized method for efficient amplicon recovery from droplets. In this work, we present a method to reliably stabilize emulsions and recover preamplified templates. We systematically compared our protocol with different destabilization methods and found an average 41% improvement in recovery efficiency. We anticipate that this standardized method will increase the consistency and reproducibility of ccfDNA/ctDNA analyses. This technique could be readily translated to other low-input or low-biomass samples, such as urine, saliva, or archived biopsy specimens.

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 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.

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.

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.

The LexA- two-hybrid (LexA-E2H) system was initially developed to study interactions between microbial proteins in an ( 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.

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.

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.

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.

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.