BioTechniques最新文献

Processing methods that do not induce shape distortion are essential for the analysis of tissue morphology. Although the use of agarose-gelatin double embedding to reduce shape distortion has been suggested, the effect of matrix concentration has not been addressed. Therefore, combinations of agarose (1-3%) and gelatin (1-10%) were evaluated using multicellular spheroids without extracellular matrix as a model of mechanical fragility. In this study, a blend of 2% agarose and 5% gelatin effectively prevented distortion. This protocol can enhance the morphological analysis of delicate tissues.

The Applied Biosystems Axiom Microbiome Array enables high-throughput detection of bacteria, archaea, viruses, protozoa, and fungi across multiple samples. However, its native software outputs are not compatible with common downstream analysis tools, requiring preprocessing. We identified a lack of open-source pipelines tailored to these outputs. To address this gap, we developed AxioParse, a Python-based pipeline built with the Dagster orchestration framework that automates data cleaning, taxonomic mapping, and formatting for downstream analysis. AxioParse reduces manual processing and generates datasets compatible with platforms such as QIIME2 and R, improving reproducibility and facilitating broader use of the Axiom Microbiome Array in microbiome research (https://github.com/Eghtesady-Lab-Bioinformatics/axioparse).

Phage display combined with biopanning is a powerful method for the discovery of therapeutic antibodies. Classic biopanning is lengthy and labor-intensive, requiring at least five days of work. The biopanning process is plagued by a bias toward binders with propagation advantages, causing a loss in diversity. We present a more efficient biopanning technique named 3in1 biopanning. Using non-denaturing elution and skipping the intermediary phage amplification steps, 3in1 biopanning performs all biopanning rounds in a single day and overcomes the inherent biopanning bias. 3in1 is five times faster than conventional biopanning and yields up to six times more unique hits with a wide range of affinities. It shortens lead discovery time by a week and reduces consumables and waste. We demonstrate that this novel biopanning technique is suitable for naive-synthetic and immune libraries against a variety of targets.

Bovine serum albumin (BSA) exhibits lot-to-lot variability, partly due to differences in fatty acid content. In this study, the structural properties of two BSA lots-fatty acid-bound and -free-were compared using electrophoretic, chromatographic, and spectroscopic techniques. No apparent differences in overall structure were detected by conventional methods, including UV absorbance spectroscopy, reducing and non-reducing SDS-PAGE, gel filtration chromatography, or agarose native gel electrophoresis. However, circular dichroism (CD) analysis of native BSA revealed small but significant differences in folded structure between the two lots, particularly in the microenvironment surrounding one of two tryptophan residues, a known fatty acid-binding region. These differences were further supported by the intrinsic fluorescence measurements. Under heat stress (73-76 °C), the two lots exhibited distinct behaviors. Native gel electrophoresis and CD spectroscopy conformational states with different patterns, including variations in aggregation propensity. These results demonstrate that, for the first time, combining CD and fluorescence spectroscopy with native electrophoresis under heat-stress conditions provides a sensitive and practical approach for detecting subtle conformational differences among BSA lots, offering a valuable tool for assessing protein quality and consistency.

Plasmid DNA remains the standard tool for mammalian transfection but carries bacterial backbone sequences that can reduce expression efficiency and raise biosafety concerns. Minicircle DNA eliminates these elements and improves expression but requires bacterial recombination systems and multi-day protocols. Here we present Self-circularized Overhang-based Unrecombined Products (SOUP), a rapid in vitro workflow for generating backbone-free circular expression cassettes directly from polymerase chain reaction (PCR) products. The method involves cassette amplification with engineered overhangs, type IIS restriction digestion, self-ligation, and RecBCD exonuclease treatment, producing circular monomers alongside dimers and concatemers in less than 24 hours. We evaluated SOUP against its parental plasmid carrying an identical green fluorescent protein (GFP) cassette in Human embryonic kidney 293 (HEK293) cells. At 24 h post-transfection, SOUP yielded a larger proportion of GFP-positive cells, while the plasmid supported higher per-cell intensity. By 56 h, SOUP showed a clear advantage, with increased mean fluorescence, higher transfection efficiency, and a productivity index more than 2.5-fold greater than the plasmid. These results demonstrate that SOUP constructs, despite their heterogeneous composition, support strong gene expression. The workflow is fast and inexpensive, making it accessible as a practical complement to plasmid vectors and a rapid prototyping tool for backbone-free DNA constructs.

Extracellular vesicles (EVs) are lipid bilayer-bound nanoparticles secreted by nearly all cells, with notable applications in intercellular communication and therapeutic delivery. However, conventional EV isolation techniques, such as ultracentrifugation and size exclusion chromatography, often face challenges like high cost, specialized equipment requirements, and low yield. In this study, we present a modified aqueous two-phase system (ATPS) as an alternative method for isolating EVs from raw bovine milk. This approach is scalable, cost-effective, and requires minimal specialized equipment, making it accessible for small laboratories. We demonstrate the efficiency of this method through the isolation and subsequent characterization of bovine milk-derived EVs, including morphological analysis, protein, and lipid quantification, and nucleic acid presence. The isolated EVs exhibited typical characteristics, morphology, specific protein markers (CD63 and TSG101), and a protein-to-lipid ratio consistent with extracellular vesicles. These findings validate the modified ATPS as a reliable and practical method for isolating milk-derived EVs, offering a promising tool for future research into their therapeutic potential and other applications.

The genetic stability of recombinant CHO cell lines producing therapeutic proteins is critical for ensuring consistent quality in biopharma-ceutical products. Southern blotting remains the gold standard for evaluating transgene integrity and stability in these cell lines. In the biopharmaceutical industry, transposon-based expression systems are widely utilized to generate highly productive and genetically stable CHO cell lines. However, evaluating transgene integration sites and integrity in such cell lines is challenging with standard Southern blotting protocols. This difficulty arises because transposon-mediated transfection often results in multiple independent integration sites in the host genome, each typically harboring a single transgene copy. Upon restriction enzyme digestion, similar-sized DNA fragments are generated, reducing resolution and complicating the separation and detection of the transgenes using standard blotting protocols. Here, we present a modified Southern blotting protocol that significantly improves the resolution of integration banding patterns by refining key steps, including purification of digested DNA prior to electrophoresis and an enhanced DNA transfer method. This protocol was successfully applied to analyze multiple transposon-derived CHO cell lines with high transgene copy numbers, enabling more precise and efficient detection of transgene integration.

Antimicrobial resistance poses a significant challenge for infection control, requiring the development of accurate and high-throughput diagnostic techniques. We expanded and optimized an existing DNA microarray platform for the molecular characterization of vancomycin-resistant Enterococcus (VRE) by incorporating resistance, virulence, species-specific, and typing markers. The enhanced microarray allows for the simultaneous analysis of up to 96 strains, providing detailed genetic profiles of clinical isolates. VRE strains from Romania and Bavaria, Germany, were analyzed, and the results were compared to those obtained using traditional typing methods, such as multilocus sequence typing (MLST). Next-generation sequencing (NGS) was used in parallel to validate the microarray findings and explore genomic relationships. The microarray revealed considerable genetic diversity and potential epidemiological linkages among isolates. A novel hexadecimal-based nomenclature system was introduced for standardized and scalable strain classification. Comparative analysis demonstrated that the array profiles provided greater discriminatory power and practical resolution than MLST. Receiver operating characteristic (ROC) curve analysis of 187 target genes in 220 isolates gave diagnostic sensitivity and specificity of 100%. This integrated approach offers a cost-effective, rapid, and adaptable global VRE surveillance and infection control tool. It provides a practical alternative to conventional typing systems and facilitates early detection of outbreaks and emerging clones.

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.