BioTechniques最新文献

{"title":"Optimization of agarose-gelatin double embedding to minimize sectioning artifacts in multicellular spheroids.","authors":"Hyoyoung Maeng, Min-Gi Han, Yoseop Jeon, Donghyeon Kim, Yuna Park, Hyuk Song","doi":"10.1080/07366205.2026.2621053","DOIUrl":"https://doi.org/10.1080/07366205.2026.2621053","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"1-5"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146103645","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation and profiling of single circulating tumor cells in myeloma: a new workflow for liquid biopsies.","authors":"Yulia Shifrin, Asieh Alikhah, Zahabiya Husain, Michelle Nguyen, Atacenk Baslik, Darryl Dyck, Silvana Ferreira, Rayan Kaedbey, Sandra Mazzoni, Sabine Mai","doi":"10.1080/07366205.2026.2645352","DOIUrl":"https://doi.org/10.1080/07366205.2026.2645352","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Minimal residual disease (MRD) is a key prognostic marker for progression-free and overall survival in multiple myeloma (MM). Existing high sensitivity assays primarily focus on tumor burden assessment, rely on bone marrow sampling, and are limited in their ability to support frequent longitudinal disease monitoring. Here, we describe a proof-of-principle workflow for isolating morphologically preserved circulating tumor cells (CTCs) from peripheral blood (PB) using size-based filtration. Based on controlled spiking experiments with RPMI 8226 myeloma cells, we demonstrate an analytical limit of detection of approximately 1 tumor cell per 10&lt;sup&gt;7&lt;/sup&gt; white blood cells. Isolated cells retain nuclear integrity and cytomorphology, allowing for downstream immuno-phenotyping, three-dimensional (3D) telomere fluorescence &lt;i&gt;in situ&lt;/i&gt; hybridization (FISH), and single-cell telomere profiling, a known marker of genomic instability and disease progression in multiple myeloma. The proposed workflow demonstrated its feasibility for isolating, profiling, and analyzing plasma cells from PB of MM patients at different disease stages. It revealed distinct nuclear and telomeric features in MM CTCs compared with normal lymphocytes. The established technically robust liquid biopsy workflow enables 3D telomere profiling of MM CTCs that can be adopted for noninvasive MRD monitoring based on genomic instability rather than on the enumeration of MM plasma cells alone.Article HighlightsCurrent high-sensitivity assays for assessing minimal residual disease (MRD) in multiple myeloma (MM) patients rely on invasive bone marrow sampling and are limited by sampling bias and poor suitability for frequent longitudinal monitoring.This study presents a proof-of-principle liquid biopsy workflow that enables isolation of morphologically intact circulating tumor cells (CTCs) from peripheral blood (PB) using size-based filtration with the ScreenCell&lt;sup&gt;®&lt;/sup&gt; device.Controlled spiking experiments with RPMI 8226 myeloma cells established an analytical limit of detection of approximately 1 tumor cell per 10&lt;sup&gt;7&lt;/sup&gt; white blood cells.Technical feasibility of the new workflow for isolating intact CTCs from liquid biopsy was confirmed in a cohort of 20 newly diagnosed MM patients at diagnosis, during induction therapy, and after relapse, supporting its potential utility for longitudinal disease monitoring.Isolated CTCs were successfully immunophenotyped and subjected to quantitative three-dimensional telomere fluorescence &lt;i&gt;in situ&lt;/i&gt; hybridization (FISH), allowing single-cell analysis of telomere length, number, aggregation, nuclear volume, and spatial distribution.Quantitative telomere profiling revealed statistically significant differences in nuclear and telomeric parameters between MM CTCs and normal lymphocytes, consistent with known markers of genomic instability and disease aggressiveness in MM.By combining enumeration with risk assessment based on telomere profi","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"123-136"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147502910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AxioParse: streamlining Axiom Microbiome assay data processing and dataset generation.","authors":"Pranav Kirti, Pirooz Eghtesady, Mathieu Garand","doi":"10.1080/07366205.2026.2644218","DOIUrl":"https://doi.org/10.1080/07366205.2026.2644218","url":null,"abstract":"<p><p>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).</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"93-97"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147472616","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"RP-HPLC-based purification of long single-stranded DNA for CRISPR knock-in applications.","authors":"Justina Martinkienė, Tingting Cui, Giovanni Ciotta, Juozas Kupčinskas, Deividas Pažėraitis","doi":"10.1080/07366205.2026.2649857","DOIUrl":"https://doi.org/10.1080/07366205.2026.2649857","url":null,"abstract":"<p><p><b>Background</b>Long single-stranded DNA (ssDNA; >200 nucleotides) is valuable for DNA nanotechnology, precision medicine, and as a CRISPR-Cas9 knock-in donor template, but existing preparation methods are laborious, low-yield, or difficult to scale. <b>Methods</b>We developed a workflow combining enzymatic digestion with high-temperature reversed-phase high-performance liquid chromatography (RP-HPLC) to purify kilobase-length ssDNA. The method was evaluated across analytical and semi-preparative formats. <b>Results</b>The approach enables clean resolution of linear and circular ssDNA species ranging from 1.5 to 4.5 kb and is scalable across formats. A 1.5 kb ssDNA donor supported efficient CRISPR knock-in at the T-cell receptor alpha constant (TRAC) locus in primary human CD8⁺ T cells, without adversely affecting viability or expansion. <b>Conclusions</b>This RP-HPLC workflow provides a scalable and reproducible method for generating high-purity long ssDNA suitable for genome engineering applications.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"149-157"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147637823","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"3in1: an ultra-fast phage display biopanning method overcoming the biopanning bias and yielding more diverse binders.","authors":"Samson Lichtenstein, Morgane Valles, Michael Mullin, Aleardo Morelli","doi":"10.1080/07366205.2026.2629753","DOIUrl":"10.1080/07366205.2026.2629753","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"41-54"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147321229","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A robust and sensitive method for detecting subtle structural differences in bovine serum albumin.","authors":"Teruo Akuta, Tomomi Sato, Masataka Nakagawa, Yuki Komatsu, Tsutomu Arakawa, Taiji Oyama","doi":"10.1080/07366205.2026.2635455","DOIUrl":"10.1080/07366205.2026.2635455","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"65-80"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147343282","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A simple and rapid agarose gel electrophoresis method to assess CpG methylation of DNA.","authors":"Marco Paliza-Carre, Ge Chen, Shahrokh Shabahang","doi":"10.1080/07366205.2026.2656194","DOIUrl":"https://doi.org/10.1080/07366205.2026.2656194","url":null,"abstract":"<p><p>The presence of 5-methylcytosine at CpG sites in mammalian DNA plays a significant role in various biological processes, both normal and aberrant. Similarly, CpG site methylation status of therapeutic DNAs can affect activity and therapeutic efficacy, as is the case for ADI-100, an immune tolerance-inducing drug candidate for the treatment of type 1 diabetes and other GAD-associated autoimmune diseases. To assess the methylation characteristics of ADI-100 and other therapeutic DNA product candidates, we developed a simple and versatile method, methylation-sensitive restriction enzyme digest and agarose gel electrophoresis (MSRE-AGE), using methylation-sensitive restriction enzyme digest, agarose gel electrophoresis, and custom <i>in silico</i> band pattern analysis for quantitative measurement of CpG methylation levels and patterns. We compared MSRE-AGE with bisulfite pyrosequencing methylation analysis of plasmid DNA and a synthetically-produced closed-linear DNA, Doggybone DNA (dbDNA), demonstrating that MSRE-AGE produces results consistent with bisulfite pyrosequencing but without structure and sequence-dependent artifacts. These findings demonstrate that MSRE-AGE is a robust and practical method for methylation analysis of therapeutic DNA products.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"170-182"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147728224","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Self-circularized Overhang-based Unrecombined Products (SOUP): a rapid in vitro method for generating backbone-free expression cassettes.","authors":"Ionuț Adrian Cepleanu-Pascu, Mirel Adrian Popa, Gheorghe Dănuț Cimponeriu, Ileana Stoica","doi":"10.1080/07366205.2026.2645351","DOIUrl":"https://doi.org/10.1080/07366205.2026.2645351","url":null,"abstract":"<p><p>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 (<b>PCR</b>) 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.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"81-92"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147455666","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation and validation of C-terminal LRP8 antibodies for detecting processed intracellular fragments.","authors":"Alessandro Medoro, Emanuele Foderà, Maurizio Ronci, Marco Trerotola, Donatella Mignogna, Gennaro Raimo, Mariano Intrieri, Anna De Martino, Alessio Lepore, Carola Porcile, Teresa Benincasa, Claudio Russo, Daniela Passarella","doi":"10.1080/07366205.2026.2645347","DOIUrl":"https://doi.org/10.1080/07366205.2026.2645347","url":null,"abstract":"<p><p><b>Introduction:</b> Low-density lipoprotein receptor-related protein 8 (LRP8) is a neuronal receptor for apolipoprotein E and Reelin, two ligands critically involved in Alzheimer's disease (AD), neuronal migration, and memory. Because LRP8 is highly expressed in neurons, interacts with amyloid precursor protein, and undergoes γ-secretase-dependent processing, it has emerged as a potential contributor to AD-related neurodegeneration. Growing evidence also implicates LRP8 in carcinogenesis, highlighting the need to better define its molecular properties. <b>Areas covered:</b> This article addresses the limited understanding of LRP8 proteolytic processing, cellular localization, and molecular interactions, due in part to the lack of suitable antibodies. We present and characterize novel polyclonal and monoclonal antibodies directed against the C-terminal region of LRP8, suitable for Western blotting and immunocytochemistry/immunofluorescence. These reagents enabled detection of a previously unrecognized intracellular low-molecular-weight (∼12 kDa) C-terminal LRP8 fragment. <b>Expert opinion/Commentary:</b> These antibodies provide valuable new tools for mechanistic studies of LRP8. By improving the investigation of LRP8 processing and localization, they may facilitate a better understanding of its role in neurodegeneration and cancer.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"111-122"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147589824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation and characterization of bovine-milk derived extracellular vesicles using a modified aqueous two-phase system.","authors":"Logan Scott Whitney, Elley Ruth College, Jonah Peña-Ekker, Charlee Ann Cannon, Kolbe Mark Mason, Jaren Nathan Wilson, Jessica E Pullan","doi":"10.1080/07366205.2026.2633107","DOIUrl":"https://doi.org/10.1080/07366205.2026.2633107","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"55-64"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147497751","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}