Pub Date : 2026-03-14DOI: 10.1016/j.ymeth.2026.03.003
Anshaal Furrukh, Herra Javed, Mohamed Refaat Ahmed Zaghw, Meagan Olivia Rogers, Simon Chung, Eli Contoro, Louis Steen, Amna Qasim, Rodolfo Henrich Lobo, Tori Beach, John D Lowery, Melaney Gee, David Irby, Taufiek Konrad Rajab
Piglets serve as an excellent model for cardiovascular research due to the anatomic and physiological similarities between the porcine and human heart. This study establishes an improved protocol for performing partial heart transplantation (PHT) in a swine model, addressing key technical and physiological challenges while optimizing perioperative management. Here we present the results of performing the PHT protocol in 70 piglets (35 donors and 35 recipients) using a standardized approach incorporating sternotomies, vascular anastomoses, and immunosuppression, and 6 unoperated controls. Intraoperative complications, primarily due to hemodynamic instability and arrhythmias, occurred in 22.9% of recipients (n = 8/35), while postoperative complications, primarily due to failure to thrive and infections occurred in 29.6% of the survivors (n = 8/27). Refinements such as prophylactic antiarrhythmic therapy, use of clamps to control bleeding in the field, and optimized selection of suture materials improved operative and post-operative outcomes over the course of the study. This protocol demonstrates the feasibility of PHT in a swine model and provides a framework for evaluating clinical partial heart transplant protocols.
{"title":"Protocol for partial heart transplantation of the pulmonary valve in a swine model: surgical technique, postoperative care, assessments and outcomes.","authors":"Anshaal Furrukh, Herra Javed, Mohamed Refaat Ahmed Zaghw, Meagan Olivia Rogers, Simon Chung, Eli Contoro, Louis Steen, Amna Qasim, Rodolfo Henrich Lobo, Tori Beach, John D Lowery, Melaney Gee, David Irby, Taufiek Konrad Rajab","doi":"10.1016/j.ymeth.2026.03.003","DOIUrl":"10.1016/j.ymeth.2026.03.003","url":null,"abstract":"<p><p>Piglets serve as an excellent model for cardiovascular research due to the anatomic and physiological similarities between the porcine and human heart. This study establishes an improved protocol for performing partial heart transplantation (PHT) in a swine model, addressing key technical and physiological challenges while optimizing perioperative management. Here we present the results of performing the PHT protocol in 70 piglets (35 donors and 35 recipients) using a standardized approach incorporating sternotomies, vascular anastomoses, and immunosuppression, and 6 unoperated controls. Intraoperative complications, primarily due to hemodynamic instability and arrhythmias, occurred in 22.9% of recipients (n = 8/35), while postoperative complications, primarily due to failure to thrive and infections occurred in 29.6% of the survivors (n = 8/27). Refinements such as prophylactic antiarrhythmic therapy, use of clamps to control bleeding in the field, and optimized selection of suture materials improved operative and post-operative outcomes over the course of the study. This protocol demonstrates the feasibility of PHT in a swine model and provides a framework for evaluating clinical partial heart transplant protocols.</p>","PeriodicalId":390,"journal":{"name":"Methods","volume":" ","pages":"87-99"},"PeriodicalIF":4.3,"publicationDate":"2026-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147466379","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-12DOI: 10.1016/j.ymeth.2026.03.004
Elisa Longo, Greta Paternò, Luca Lanzanò
Lower excitation light levels reduce phototoxicity in confocal and superresolution microscopy but generate images with lower photon counts. A well-established parameter that quantifies chromatin compaction from fluorescence images of labeled nuclei is the coefficient of variation (CV), which is defined as the ratio between the standard deviation and the average of the pixel intensity values. Here, we show that when imaging is performed at low photon counts the standard deviation of the intensity is dominated by the shot noise fluctuations and the CV is not representative of the chromatin compaction state. To overcome this issue, we introduce an alternative calculation of CV based on image correlation spectroscopy (CV-ICS), where the CV-ICS parameter is extracted from the spatial autocorrelation function of the image. We demonstrate the use of CV-ICS on fast confocal (resonant) imaging of live HeLa cells labeled with Hoechst 33342. First, we show that the conventional CV calculation deviates already by about 50% from the correct value when the maximum photon counts per pixel are below 40. In contrast, the CV-ICS calculation is unaffected by the presence of shot noise. Next, we use imaging at low photon counts and CV-ICS to follow changes in the chromatin compaction state after switching the cells to a medium of higher (hyper-) or lower (hypo-) osmolarity. Finally, we use CV-ICS to investigate the time course of chromatin compaction after induction of laser-induced DNA damage. This work shows that CV-ICS can be a robust parameter for quantifying chromatin compaction in all imaging applications with a limited photon budget, including live cell imaging and superresolution microscopy.
{"title":"The coefficient of variation by image correlation spectroscopy (CV-ICS) quantifies chromatin compaction from images with low photon counts.","authors":"Elisa Longo, Greta Paternò, Luca Lanzanò","doi":"10.1016/j.ymeth.2026.03.004","DOIUrl":"10.1016/j.ymeth.2026.03.004","url":null,"abstract":"<p><p>Lower excitation light levels reduce phototoxicity in confocal and superresolution microscopy but generate images with lower photon counts. A well-established parameter that quantifies chromatin compaction from fluorescence images of labeled nuclei is the coefficient of variation (CV), which is defined as the ratio between the standard deviation and the average of the pixel intensity values. Here, we show that when imaging is performed at low photon counts the standard deviation of the intensity is dominated by the shot noise fluctuations and the CV is not representative of the chromatin compaction state. To overcome this issue, we introduce an alternative calculation of CV based on image correlation spectroscopy (CV-ICS), where the CV-ICS parameter is extracted from the spatial autocorrelation function of the image. We demonstrate the use of CV-ICS on fast confocal (resonant) imaging of live HeLa cells labeled with Hoechst 33342. First, we show that the conventional CV calculation deviates already by about 50% from the correct value when the maximum photon counts per pixel are below 40. In contrast, the CV-ICS calculation is unaffected by the presence of shot noise. Next, we use imaging at low photon counts and CV-ICS to follow changes in the chromatin compaction state after switching the cells to a medium of higher (hyper-) or lower (hypo-) osmolarity. Finally, we use CV-ICS to investigate the time course of chromatin compaction after induction of laser-induced DNA damage. This work shows that CV-ICS can be a robust parameter for quantifying chromatin compaction in all imaging applications with a limited photon budget, including live cell imaging and superresolution microscopy.</p>","PeriodicalId":390,"journal":{"name":"Methods","volume":" ","pages":"55-64"},"PeriodicalIF":4.3,"publicationDate":"2026-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147455181","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-12-25DOI: 10.1016/j.ymeth.2025.12.011
Ok Kim , Kyung-Hwa Choi , Tae Hui Kim , Sungmin Son , Jonghun Lee , Kyungmin Kim , Un Sun Chung , Eun-Jin Cheon , Ilju Lee , Hyunwoo Jung , Ho-Jang Kwon , Xue Han , Jonghyuk Choi , Jung Won Kim , Ah Lahm Shin , Jung Jae Lee
The Korean Longitudinal Study on Digitally Optimized Mental Healthcare is an innovative multicenter trial-ready cohort study. It aims to develop a digitally integrated mental healthcare platform that integrates robots, artificial intelligence, and local community services. A total of 3,100 participants, including 1,000 from the previous Chungnam Province cohort and 700 each from Chungnam, Gangwon, and Daegu, will be recruited between December 2024 and the end of 2027. Sociodemographic factors and physical health data are collected at enrollment using questionnaires. Every four months, formal tools are used to conduct psychiatric diagnoses and determine participants’ mental health condition, such as depressive symptoms, anxiety symptoms, suicidality, stress, insomnia, loneliness, quality of life, and disability. We assessed smartphone overdependence at regular intervals. Passive data on phone usage, life patterns, and health monitoring from a smart band are collected in real time. Weekly ecological momentary assessments monitor daily life and mood and detect risk situations early. A subset of 500 participants engages in a Living Lab, collecting multi-modal data through robots and sleep radar sensors while evaluating the usability of and satisfaction with these devices. This study will show how tailored digital applications and web-based platforms can facilitate personalized self-help interventions, enhance expert–user interaction, and promote active user engagement. This approach can potentially reduce stigma and improve public awareness of mental healthcare by shifting from a treatment-centered model to a community-based prevention framework.
{"title":"Korean longitudinal study on digitally optimized mental healthcare: a cohort profile","authors":"Ok Kim , Kyung-Hwa Choi , Tae Hui Kim , Sungmin Son , Jonghun Lee , Kyungmin Kim , Un Sun Chung , Eun-Jin Cheon , Ilju Lee , Hyunwoo Jung , Ho-Jang Kwon , Xue Han , Jonghyuk Choi , Jung Won Kim , Ah Lahm Shin , Jung Jae Lee","doi":"10.1016/j.ymeth.2025.12.011","DOIUrl":"10.1016/j.ymeth.2025.12.011","url":null,"abstract":"<div><div>The Korean Longitudinal Study on Digitally Optimized Mental Healthcare is an innovative multicenter trial-ready cohort study. It aims to develop a digitally integrated mental healthcare platform that integrates robots, artificial intelligence, and local community services. A total of 3,100 participants, including 1,000 from the previous Chungnam Province cohort and 700 each from Chungnam, Gangwon, and Daegu, will be recruited between December 2024 and the end of 2027. Sociodemographic factors and physical health data are collected at enrollment using questionnaires. Every four months, formal tools are used to conduct psychiatric diagnoses and determine participants’ mental health condition, such as depressive symptoms, anxiety symptoms, suicidality, stress, insomnia, loneliness, quality of life, and disability. We assessed smartphone overdependence at regular intervals. Passive data on phone usage, life patterns, and health monitoring from a smart band are collected in real time. Weekly ecological momentary assessments monitor daily life and mood and detect risk situations early. A subset of 500 participants engages in a Living Lab, collecting multi-modal data through robots and sleep radar sensors while evaluating the usability of and satisfaction with these devices. This study will show how tailored digital applications and web-based platforms can facilitate personalized self-help interventions, enhance expert–user interaction, and promote active user engagement. This approach can potentially reduce stigma and improve public awareness of mental healthcare by shifting from a treatment-centered model to a community-based prevention framework.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 41-49"},"PeriodicalIF":4.3,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145846209","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-12-18DOI: 10.1016/j.ymeth.2025.12.006
Tanya Lupancu , Jonathan Limpah , Esrin Aydin , Timothy Fan , Vanessa Wong , Joanne Rimmer , Brian S. Wang , David M. Yen , Eldin Rostom , Adam M. Damry
Nasal fluid biomarker analysis is an emerging technique for studying sinonasal pathophysiology, monitoring therapeutic efficacy, and discovering novel drug targets. Variability in biomarker results can be contributed to non-standardized collection methodology. To address this, a novel microsampler was developed, designed to enable precise site-specific sampling, consistent volume collection, and high analyte recovery. This study aims to evaluate the performance of this new microsampler device compared to commonly utilized flocked swab, and other absorbent materials. To do so, fixed volumes of a synthetic nasal fluid mimic were deposited onto the anterior region of the inferior turbinate of a 3D-printed sinus model to assess volumetric and collection site accuracy of the nasal microsampler, in comparison to a flocked swab. Additionally, protein biomarker recovery properties of the device’s absorption membrane, LeukosorbTM, versus experimental proprietary absorbent materials, were assessed using ELISA. The microsampler, contrasting the flocked swab, demonstrated statistically significant lower coefficient of variation for collected nasal fluid volume and greater sampling site precision. The spike and recovery study indicated that the proprietary materials had statistically significant higher biomarker recovery rates than LeukosorbTM. Overall, the novel nasal microsampler offers significantly improved volumetric control and site-specific collection against flocked swab. All experimental proprietary absorbent materials displayed significantly higher protein recovery rates, comparing to widely accepted and utilized LeukosorbTM. Consistent use of the novel nasal microsampler device has the potential to standardize protein recovery processes and minimize variability across studies, leading to enhanced reliability and comparability of future findings.
{"title":"Development of a novel microsampling device to standardize the analysis of intranasal inflammatory biomarkers","authors":"Tanya Lupancu , Jonathan Limpah , Esrin Aydin , Timothy Fan , Vanessa Wong , Joanne Rimmer , Brian S. Wang , David M. Yen , Eldin Rostom , Adam M. Damry","doi":"10.1016/j.ymeth.2025.12.006","DOIUrl":"10.1016/j.ymeth.2025.12.006","url":null,"abstract":"<div><div>Nasal fluid biomarker analysis is an emerging technique for studying sinonasal pathophysiology, monitoring therapeutic efficacy, and discovering novel drug targets. Variability in biomarker results can be contributed to non-standardized collection methodology. To address this, a novel microsampler was developed, designed to enable precise site-specific sampling, consistent volume collection, and high analyte recovery. This study aims to evaluate the performance of this new microsampler device compared to commonly utilized flocked swab, and other absorbent materials. To do so, fixed volumes of a synthetic nasal fluid mimic were deposited onto the anterior region of the inferior turbinate of a 3D-printed sinus model to assess volumetric and collection site accuracy of the nasal microsampler, in comparison to a flocked swab. Additionally, protein biomarker recovery properties of the device’s absorption membrane, Leukosorb<sup>TM</sup>, versus experimental proprietary absorbent materials, were assessed using ELISA. The microsampler, contrasting the flocked swab, demonstrated statistically significant lower coefficient of variation for collected nasal fluid volume and greater sampling site precision. The spike and recovery study indicated that the proprietary materials had statistically significant higher biomarker recovery rates than Leukosorb<sup>TM</sup>. Overall, the novel nasal microsampler offers significantly improved volumetric control and site-specific collection against flocked swab. All experimental proprietary absorbent materials displayed significantly higher protein recovery rates, comparing to widely accepted and utilized Leukosorb<sup>TM</sup>. Consistent use of the novel nasal microsampler device has the potential to standardize protein recovery processes and minimize variability across studies, leading to enhanced reliability and comparability of future findings.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 25-33"},"PeriodicalIF":4.3,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145800355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
POT1 is the only single stranded telomere binding protein in the shelterin complex. Together with TPP1, POT1 plays a crucial role in regulating telomere length and protecting telomeres from DNA damage repair proteins. The activation of DNA damage repair proteins at telomeres can be detrimental to cells, so their activity must be suppressed. POT1 interacts with other telomeric proteins (TRF2, TRF1, TIN2 and RAP1) via its association with TPP1. These proteins function together to protect and maintain the telomeres. Despite extensive knowledge of POT1′s role within the shelterin complex, the full spectrum of its interactors at the single-stranded telomeric overhang remains poorly defined. To study these interactions, we generated an endogenous Flag-tag knock-in of POT1 using the CRISPR-Cas9 gene editing system. To address the risk of unintended gene disruption associated with this technique, we conducted an in-depth characterization of the endogenously Flag-tagged POT1 clone to ensure that its telomere and TPP1 binding functions remained intact. Further, we performed proteomic profiling of the Flag-tagged POT1 within the chromatin fraction using ChIP-MS to explore its proteome. Our analysis uncovered a novel set of POT1-associated proteins at the extremes of telomeres. Given that POT1 exclusively binds to the single-stranded 3′ overhang of telomeres, the proteomic data obtained indicates POT1 interactions occurring at the extreme ends of telomeres. In conclusion, our study reveals previously uncharacterized POT1 associated proteins using ChIP mass spectrometric approach, paving the way for further investigations into telomere biology and potential therapies targeting telomere regulation.
{"title":"Proteomics method for identifying POT1-associated complexes at telomeres using ChIP-Mass spectrometry","authors":"Nikita Harish Zade , Meghna Jain , Manoj Garg , Rahul Checker , Arkasubhra Ghosh , Ekta Khattar","doi":"10.1016/j.ymeth.2026.01.007","DOIUrl":"10.1016/j.ymeth.2026.01.007","url":null,"abstract":"<div><div>POT1 is the only single stranded telomere binding protein in the shelterin complex. Together with TPP1, POT1 plays a crucial role in regulating telomere length and protecting telomeres from DNA damage repair proteins. The activation of DNA damage repair proteins at telomeres can be detrimental to cells, so their activity must be suppressed. POT1 interacts with other telomeric proteins (TRF2, TRF1, TIN2 and RAP1) via its association with TPP1. These proteins function together to protect and maintain the telomeres. Despite extensive knowledge of POT1′s role within the shelterin complex, the full spectrum of its interactors at the single-stranded telomeric overhang remains poorly defined. To study these interactions, we generated an endogenous Flag-tag knock-in of POT1 using the CRISPR-Cas9 gene editing system. To address the risk of unintended gene disruption associated with this technique, we conducted an in-depth characterization of the endogenously Flag-tagged POT1 clone to ensure that its telomere and TPP1 binding functions remained intact. Further, we performed proteomic profiling of the Flag-tagged POT1 within the chromatin fraction using ChIP-MS to explore its proteome. Our analysis uncovered a novel set of POT1-associated proteins at the extremes of telomeres. Given that POT1 exclusively binds to the single-stranded 3′ overhang of telomeres, the proteomic data obtained indicates POT1 interactions occurring at the extreme ends of telomeres. In conclusion, our study reveals previously uncharacterized POT1 associated proteins using ChIP mass spectrometric approach, paving the way for further investigations into telomere biology and potential therapies targeting telomere regulation.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 161-174"},"PeriodicalIF":4.3,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146040153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2026-01-07DOI: 10.1016/j.ymeth.2026.01.003
Alyssa N. Gonneville , Alyssa E. Ward , Narisa Ria Naidoo , Francisco N. Barrera , Rajan Lamichhane
Single-molecule fluorescence experiments are a powerful tool for studying biomolecular interactions, including protein dynamics and oligomerization, protein–protein interactions, and protein-nucleic acid interactions. Biomolecules are commonly immobilized on the microscope surface to extend the observation time. However, non-specific interactions between biomolecules and the surface present a major challenge. The first critical step in these experiments is preparing the surface using polyethylene glycol (PEG) coated slides, which facilitate biomolecule immobilization while minimizing non-specific interactions. The surface treatment typically uses PEG-SVA (Succinimidyl Valerate) coated slides, and the protocol for the treatment is lengthy and time-consuming. To overcome this issue, we have developed a process that uses PEG-Silane to improve efficiency while maintaining reproducibility. Here, we present a one-step, rapid PEGylation methodology that can be completed in minutes rather than hours. We demonstrate its validity and feasibility through single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule photobleaching experiments across various biological samples.
{"title":"Rapid and improved surface passivation method for Single-Molecule experiments","authors":"Alyssa N. Gonneville , Alyssa E. Ward , Narisa Ria Naidoo , Francisco N. Barrera , Rajan Lamichhane","doi":"10.1016/j.ymeth.2026.01.003","DOIUrl":"10.1016/j.ymeth.2026.01.003","url":null,"abstract":"<div><div>Single-molecule fluorescence experiments are a powerful tool for studying biomolecular interactions, including protein dynamics and oligomerization, protein–protein interactions, and protein-nucleic acid interactions. Biomolecules are commonly immobilized on the microscope surface to extend the observation time. However, non-specific interactions between biomolecules and the surface present a major challenge. The first critical step in these experiments is preparing the surface using polyethylene glycol (PEG) coated slides, which facilitate biomolecule immobilization while minimizing non-specific interactions. The surface treatment typically uses PEG-SVA (Succinimidyl Valerate) coated slides, and the protocol for the treatment is lengthy and time-consuming. To overcome this issue, we have developed a process that uses PEG-Silane to improve efficiency while maintaining reproducibility. <em>Here</em>, we present a one-step, rapid PEGylation methodology that can be completed in minutes rather than hours. We demonstrate its validity and feasibility through single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule photobleaching experiments across various biological samples.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 95-106"},"PeriodicalIF":4.3,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145942009","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sequence-specific binding is at the core of all DNA-templated processes, including the initiation of DNA replication, gene expression and DNA repair. Yet the kinetics and precision of these interactions remain difficult to capture at high resolution. Here, we present Dynamic UV Laser Footprinting (DULF), a novel technique that integrates UV laser footprinting with stopped-flow mixing to probe transcription factor (TF)-DNA interactions at millisecond temporal and single base-pair spatial resolution. Using NF-κB as a model TF, we demonstrate the feasibility of DULF in visualizing the spatiotemporal details of a sequence-specific TF-DNA interaction. The high temporal resolution of our data reveals that TF-DNA binding proceeds through a rapid recognition step and a subsequent slower stabilization phase. DULF also revealed that the p50 homodimer binds specifically to DNA outside the canonical binding site, emphasizing the role of flanking sequences in these interactions. All-atom molecular dynamics simulations confirmed that DNA sequence context, including flanking base pairs, modulates NF-κB binding stability and induces local structural changes such as bending, groove widening, and base unstacking. DULF offers a unique opportunity to study DNA-protein interactions at unprecedented resolution, providing insights into the mechanism of sequence-specific binding and stabilization of chromatin interactors.
{"title":"Precision mapping of NF-κB-DNA binding: high-resolution insights via dynamic UV-laser footprinting","authors":"Imtiaz Nisar Lone , Gözdem Çavdar , Zahari Peshev , Seyit Kale , Dimitar Angelov","doi":"10.1016/j.ymeth.2026.01.005","DOIUrl":"10.1016/j.ymeth.2026.01.005","url":null,"abstract":"<div><div>Sequence-specific binding is at the core of all DNA-templated processes, including the initiation of DNA replication, gene expression and DNA repair. Yet the kinetics and precision of these interactions remain difficult to capture at high resolution. <em>Here</em>, we present Dynamic UV Laser Footprinting (DULF), a novel technique that integrates UV laser footprinting with stopped-flow mixing to probe transcription factor (TF)-DNA interactions at millisecond temporal and single base-pair spatial resolution. Using NF-κB as a model TF, we demonstrate the feasibility of DULF in visualizing the spatiotemporal details of a sequence-specific TF-DNA interaction. The high temporal resolution of our data reveals that TF-DNA binding proceeds through a rapid recognition step and a subsequent slower stabilization phase. DULF also revealed that the p50 homodimer binds specifically to DNA outside the canonical binding site, emphasizing the role of flanking sequences in these interactions. All-atom molecular dynamics simulations confirmed that DNA sequence context, including flanking base pairs, modulates NF-κB binding stability and induces local structural changes such as bending, groove widening, and base unstacking. DULF offers a unique opportunity to study DNA-protein interactions at unprecedented resolution, providing insights into the mechanism of sequence-specific binding and stabilization of chromatin interactors.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 50-62"},"PeriodicalIF":4.3,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145922632","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2026-01-05DOI: 10.1016/j.ymeth.2026.01.001
Jihye Park , Seoyeon Youn , Kyuho Kang , Keunsoo Kang
RNA sequencing datasets in the gene expression omnibus (GEO) increasingly include NCBI-generated count matrices, enabling streamlined signature gene discovery. We present ERAPID, a computational framework that automatically processes this public data for robust differential expression analysis. The pipeline integrates metadata harmonization, surrogate variable analysis (SVA) to capture latent technical variation for batch correction, dual differential expression methods (DESeq2 and dream), gene set enrichment analysis (GSEA), and evidence-based gene prioritization via automated literature mining. ERAPID delivers interactive dashboards (volcano/MA plots, heatmaps, enrichment reports, searchable DEG tables) and supports an optional meta‑analysis step. Applied to a neuropsychiatric cohort (GSE80655), ERAPID completed analysis on a standard laptop in under an hour, recapitulating the reported association of EGR1 with schizophrenia. In an Alzheimer’s disease (AD) case study integrating four GEO datasets, ERAPID identified 17 DEGs consistently altered across all AD‑versus‑control comparisons (e.g., ADCYAP1, PPEF1, VGF, and CRH), with KEGG Alzheimer’s disease and oxidative phosphorylation pathways showing negative enrichment. Thus, ERAPID lowers the barrier to reusing public transcriptomes for signature gene discovery and biological interpretation.
{"title":"ERAPID: an end‑to‑end RNA‑seq analysis pipeline for integrative candidate biomarker discovery with applications to neuropsychiatric disorders","authors":"Jihye Park , Seoyeon Youn , Kyuho Kang , Keunsoo Kang","doi":"10.1016/j.ymeth.2026.01.001","DOIUrl":"10.1016/j.ymeth.2026.01.001","url":null,"abstract":"<div><div>RNA sequencing datasets in the gene expression omnibus (GEO) increasingly include NCBI-generated count matrices, enabling streamlined signature gene discovery. We present ERAPID, a computational framework that automatically processes this public data for robust differential expression analysis. The pipeline integrates metadata harmonization, surrogate variable analysis (SVA) to capture latent technical variation for batch correction, dual differential expression methods (DESeq2 and dream), gene set enrichment analysis (GSEA), and evidence-based gene prioritization via automated literature mining. ERAPID delivers interactive dashboards (volcano/MA plots, heatmaps, enrichment reports, searchable DEG tables) and supports an optional meta‑analysis step. Applied to a neuropsychiatric cohort (GSE80655), ERAPID completed analysis on a standard laptop in under an hour, recapitulating the reported association of <em>EGR1</em> with schizophrenia. In an Alzheimer’s disease (AD) case study integrating four GEO datasets, ERAPID identified 17 DEGs consistently altered across all AD‑versus‑control comparisons (e.g., <em>ADCYAP1</em>, <em>PPEF1</em>, <em>VGF</em>, and <em>CRH</em>), with KEGG Alzheimer’s disease and oxidative phosphorylation pathways showing negative enrichment. Thus, ERAPID lowers the barrier to reusing public transcriptomes for signature gene discovery and biological interpretation.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 34-40"},"PeriodicalIF":4.3,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145916601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2026-01-10DOI: 10.1016/j.ymeth.2026.01.006
Wael Sheet Hussein , Ali Fawzi Al-Hussainy , Soumya V. Menon , Laxmidhar Maharana , A. Sabarivani , Gunjan Mukherjee , Aashna Sinha , Khabilov Nigman , Hayder Naji Sameer , Rasim M. Salih , Mohaned Adil , Saman Kalantari
Nanoparticles (NPs) are intentionally incorporated into dental products to enhance material quality. Recently, metal–organic framework (MOF) NPs have attracted significant attention owing to their unique characteristics absent in their larger-scale counterparts. These properties make MOFs suitable for various functional platforms. MOF-based nanomaterials exhibit high antibacterial potential, owing to their biocompatibility, biodegradability, and safety for living organisms. They have also become a prominent component in the development of anticancer agents and in the manufacture of commercial medical products. Consequently, MOFs are becoming increasingly important in dentistry. They show promising therapeutic potential for inhibiting oral infectious diseases, such as those caused by bacterial plaque, and have a broad range of other biomedical applications, including the diagnosis and treatment of dental diseases. However, significant challenges hinder their transition from laboratory testing to widespread clinical use in hospitals. Key issues include long-term toxicity and biocompatibility concerns, unpredictable pharmacokinetics that can lead to unintended accumulation in organs like the liver and spleen, and the potential for adverse immune reactions. Other major obstacles are particle aggregation in biological fluids, the complex formation of a “protein corona” that can alter the NPs’ intended function, and the high cost of large-scale production under consistent Good Manufacturing Practices (GMP). This article discusses MOFs and their potential applications in identifying cancer biomarkers and treating tooth infections and oral cancers. It highlights their unique porous features, which help combat oral biofilms and detect microorganisms. The summary also addresses the primary clinical challenges that must be addressed to ensure the safe development of MOF-based therapies.
{"title":"The clinical potential and limitations of metal-organic frameworks (MOFs) in dentistry","authors":"Wael Sheet Hussein , Ali Fawzi Al-Hussainy , Soumya V. Menon , Laxmidhar Maharana , A. Sabarivani , Gunjan Mukherjee , Aashna Sinha , Khabilov Nigman , Hayder Naji Sameer , Rasim M. Salih , Mohaned Adil , Saman Kalantari","doi":"10.1016/j.ymeth.2026.01.006","DOIUrl":"10.1016/j.ymeth.2026.01.006","url":null,"abstract":"<div><div>Nanoparticles (NPs) are intentionally incorporated into dental products to enhance material quality. Recently, metal–organic framework (MOF) NPs have attracted significant attention owing to their unique characteristics absent in their larger-scale counterparts. These properties make MOFs suitable for various functional platforms. MOF-based nanomaterials exhibit high antibacterial potential, owing to their biocompatibility, biodegradability, and safety for living organisms. They have also become a prominent component in the development of anticancer agents and in the manufacture of commercial medical products. Consequently, MOFs are becoming increasingly important in dentistry. They show promising therapeutic potential for inhibiting oral infectious diseases, such as those caused by bacterial plaque, and have a broad range of other biomedical applications, including the diagnosis and treatment of dental diseases. However, significant challenges hinder their transition from laboratory testing to widespread clinical use in hospitals. Key issues include long-term toxicity and biocompatibility concerns, unpredictable pharmacokinetics that can lead to unintended accumulation in organs like the liver and spleen, and the potential for adverse immune reactions. Other major obstacles are particle aggregation in biological fluids, the complex formation of a “protein corona” that can alter the NPs’ intended function, and the high cost of large-scale production under consistent Good Manufacturing Practices (GMP). This article discusses MOFs and their potential applications in identifying cancer biomarkers and treating tooth infections and oral cancers. It highlights their unique porous features, which help combat oral biofilms and detect microorganisms. The summary also addresses the primary clinical challenges that must be addressed to ensure the safe development of MOF-based therapies.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 107-131"},"PeriodicalIF":4.3,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145958439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2026-01-08DOI: 10.1016/j.ymeth.2026.01.002
Pankaj Pal , Monika Sharma , Sukesh Kumar Gupta
The expanding field of neuroscience has increasingly recognized the critical role of microglia, the resident immune cells of the central nervous system (CNS), in human health and disease. This review embarks on an in-depth exploration of the current landscape of human microglia extraction techniques. With a focus on the unique challenges and opportunities presented by human tissue samples, we delve into various sources of human microglia, including primary brain tissue and stem cell-derived models, addressing the ethical and logistical considerations inherent to these methodologies. Laboratory techniques for microglia extraction are scrutinized, highlighting adaptations for human samples in enzymatic digestion, mechanical dissociation, density gradient centrifugation, and cell sorting, alongside innovations in cryopreservation and viability assessment. Critical perspectives are offered on technical hurdles, technique selection for human research, and the horizon of emerging technologies. Standardization efforts and international guidelines are discussed, underscoring their significance in fostering reproducibility and comparability across studies. The review also illuminates applications of human microglia in disease modeling, drug discovery, and understanding neurodevelopmental and neurodegenerative processes. Concluding remarks emphasize future directions of human microglia research, advocating for methodological precision and integration of next-generation technologies to unlock new therapeutic avenues. This review serves as a comprehensive guide for researchers navigating the complex terrain of human microglia extraction, aiming to catalyze advancements in neuroscience research through methodological innovation and standardization.
{"title":"Navigating the frontier of human microglia isolation—standardization and translational insight","authors":"Pankaj Pal , Monika Sharma , Sukesh Kumar Gupta","doi":"10.1016/j.ymeth.2026.01.002","DOIUrl":"10.1016/j.ymeth.2026.01.002","url":null,"abstract":"<div><div>The expanding field of neuroscience has increasingly recognized the critical role of microglia, the resident immune cells of the central nervous system (CNS), in human health and disease. This review embarks on an in-depth exploration of the current landscape of human microglia extraction techniques. With a focus on the unique challenges and opportunities presented by human tissue samples, we delve into various sources of human microglia, including primary brain tissue and stem cell-derived models, addressing the ethical and logistical considerations inherent to these methodologies. Laboratory techniques for microglia extraction are scrutinized, highlighting adaptations for human samples in enzymatic digestion, mechanical dissociation, density gradient centrifugation, and cell sorting, alongside innovations in cryopreservation and viability assessment. Critical perspectives are offered on technical hurdles, technique selection for human research, and the horizon of emerging technologies. Standardization efforts and international guidelines are discussed, underscoring their significance in fostering reproducibility and comparability across studies. The review also illuminates applications of human microglia in disease modeling, drug discovery, and understanding neurodevelopmental and neurodegenerative processes. Concluding remarks emphasize future directions of human microglia research, advocating for methodological precision and integration of next-generation technologies to unlock new therapeutic avenues. This review serves as a comprehensive guide for researchers navigating the complex terrain of human microglia extraction, aiming to catalyze advancements in neuroscience research through methodological innovation and standardization.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 63-84"},"PeriodicalIF":4.3,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145922633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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