Methods最新文献

{"title":"Site-specific protein conjugates incorporating Para-Azido-L-Phenylalanine for cellular and in vivo imaging","authors":"Hailey E. Lightle,&nbsp;Parmila Kafley,&nbsp;Todd R. Lewis,&nbsp;Rongsheng E. Wang","doi":"10.1016/j.ymeth.2023.10.001","DOIUrl":"10.1016/j.ymeth.2023.10.001","url":null,"abstract":"<div><p>This work features the use of amber suppression-mediated unnatural amino acid (UAA) incorporation into proteins for various imaging purposes. The site-specific incorporation of the UAA, p-azido-L-phenylalanine (pAzF), provides an azide handle that can be used to complete the strain promoted azide-alkyne click cycloaddition (SPAAC) reaction to introduce an imaging modality such as a fluorophore or a positron emission tomography (PET) tracer on the protein of interest (POI). Such methodology can be pursued directly in mammalian cell lines or on proteins expressed <em>in vitro</em>, thereby conferring a homogeneous pool of protein conjugates. A general procedure for UAA incorporation to use with a site-specific protein labeling method is provided allowing for <em>in vitro</em> and <em>in vivo</em> imaging applications based on the representative proteins PTEN and PD-L1. This approach would help elucidate the cellular or <em>in vivo</em> biological activities of the POI.</p></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"219 ","pages":"Pages 95-101"},"PeriodicalIF":4.8,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41093626","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}

{"title":"A cell image segmentation method based on edge feature residual fusion","authors":"Jinlian Du,&nbsp;Yanqiu Zhang,&nbsp;Xueyun Jin,&nbsp;Xiao Zhang","doi":"10.1016/j.ymeth.2023.09.009","DOIUrl":"10.1016/j.ymeth.2023.09.009","url":null,"abstract":"<div><p>In recent years, cancer has seriously damaged human health, and the morphological structure of cells serves as an important basis for cancer diagnosis and grading. Automatic cell segmentation based on deep learning has become an important means of computer-aided pathological diagnosis. Aiming at the existing problems of rough segmentation boundaries and inaccurate segmentation in cell image segmentation, this paper designs a cell image segmentation network model (ERF-TransUNet) based on edge feature residual fusion from the perspective of mutual complementarity and constraint between edge features and object features. The model uses a hybrid architecture of CNN and Transformer to extract multi-scale features from cell images, and adds independent edge feature extraction modules and residual fusion modules to enhance the extraction of edge features and their constraints when fusing with cell object features, improving the accuracy of cell contour positioning. Through experiments on two gland cell datasets, CRAG and Glas, and comparing the segmentation effects with current popular deep learning models, the network model proposed in this paper has achieved good performance in both Dice coefficient and Hausdorff distance, which can effectively improve the segmentation effect of cell images.</p></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"219 ","pages":"Pages 111-118"},"PeriodicalIF":4.8,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046202323001639/pdfft?md5=ecf65e09185af626a206f62bc046fc3c&pid=1-s2.0-S1046202323001639-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41099541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inner leaflet cationic lipid increases nucleic acid loading independently of outer leaflet lipid charge in asymmetric liposomes","authors":"Bingchen Li ,&nbsp;Erwin London","doi":"10.1016/j.ymeth.2023.08.015","DOIUrl":"10.1016/j.ymeth.2023.08.015","url":null,"abstract":"<div><p>Use of cationic lipid vesicles (liposomes) can yield large amounts of nucleic acid entrapped inside the vesicles and/or bound to the external surface of the vesicles. To show a method to prepare asymmetric lipid vesicles (liposomes) with high amounts of entrapped nucleic acid is possible, symmetric and asymmetric lipid vesicles composed of mixtures of neutral (zwitterionic), anionic, and/or cationic phospholipids were formed in the presence of oligo DNA. For symmetric large unilamellar vesicles nucleic acid association with vesicles was roughly 100 times greater for vesicles with a net cationic charge than for vesicles having a net neutral or anionic net charge. A high degree of association between nucleic acid and lipid was also achieved using asymmetric large unilamellar vesicles with a net cationic charge in their inner leaflet, even when they had an anionic charge in their outer leaflet. In contrast, asymmetric vesicles in which only the outer leaflet had a net cationic charge had only low amounts of vesicle-associated nucleic acid, similar in amount to the amount of nucleic acid associated with asymmetric vesicles with an outer leaflet having a net anionic charge. These results indicate that in asymmetric vesicles with cationic lipid enriched inner leaflets nucleic acid is largely entrapped inside the vesicle lumen rather than bound to their external surface, and that asymmetric vesicles can be used to trap high amounts of nucleic acid even when using a lipid composition in the outer leaflet of a lipid vesicle that does not associate with nucleic acids. Such asymmetrically charged vesicles should have applications in studies of membrane protein-nucleic acid interactions as well as in studies of how membrane charge asymmetry can influence membrane protein structure, orientation, and function.</p></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"219 ","pages":"Pages 16-21"},"PeriodicalIF":4.8,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10271905","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}

{"title":"Dry blood spot samples to monitor immune-associated mRNA expression in intervention studies: Impact of Baker’s yeast beta glucan","authors":"Brian K. McFarlin ,&nbsp;Elizabeth A. Bridgeman ,&nbsp;Jakob L. Vingren ,&nbsp;David W. Hill","doi":"10.1016/j.ymeth.2023.09.006","DOIUrl":"10.1016/j.ymeth.2023.09.006","url":null,"abstract":"<div><p>Monitoring immunological response to physical stressors in a field setting is challenging because existing methods require a laboratory visit and traditional blood collection via venipuncture. The purpose of this study was to determine if our optimized dry blood spot (DBS) methodology yields sufficient total RNA to quantify the effect of Baker’s Yeast Beta Glucan supplementation (BYBG; Wellmune; 250 mg/d) on post-exercise mRNA expression. Participants had venous DBS samples collected prior to (PRE), and immediately (POST), 2 (2H), and 4 (4H) hrs after completion of a 90 min run/walk trial in a hot, humid environment. Total RNA extracted from DBS was analyzed using a 574-plex Human Immunology mRNA panel (Nanostring). BYBG supplementation was associated with the increased expression of 12 mRNAs (LTB4R, PML, PRFM1, TNFRSF14, LCK, MYD88, STAT3, CCR1, TNFSF10, LILRB3, MME, and STAT6) and decreased expression of 4 mRNAs (MAP4K1, IKBKG, CD5, and IL4R) across all post-exercise time points. In addition to individually changed mRNA targets, we found eleven immune-response pathways that were significantly enriched by BYBG following exercise (TNF Family signaling, immunometabolism, oxidative stress, toll-like receptor (TLR) signaling, Treg differentiation, autophagy, chemokine signaling, complement system, Th2 differentiation, cytokine signaling, and innate immune). The present approach showed that DBS samples can be used to yield useful information about mRNA biomarkers in an intervention study. We have found that BYBG supplementation induces changes at the mRNA level that support the immune system and reduce susceptibility to opportunistic infection (i.e., upper respiratory tract infection) and facilitate improved physical recovery from exercise. Future studies may look to use DBS sampling for testing other nutritional, health, or medical interventions.</p></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"219 ","pages":"Pages 39-47"},"PeriodicalIF":4.8,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046202323001603/pdfft?md5=d597bb6b7fb261adb15c8c1dbf34b421&pid=1-s2.0-S1046202323001603-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41096530","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PlateletSeq: A novel method for discovery of blood-based biomarkers","authors":"Ryan J. Collinson ,&nbsp;Darren Boey ,&nbsp;Lynne Wilson ,&nbsp;Zi Yun Ng ,&nbsp;Bob Mirzai ,&nbsp;Hun Chuah ,&nbsp;Michael F. Leahy ,&nbsp;Rebecca Howman ,&nbsp;Matthew Linden ,&nbsp;Kathy Fuller ,&nbsp;Wendy N. Erber ,&nbsp;Belinda B. Guo","doi":"10.1016/j.ymeth.2023.10.003","DOIUrl":"10.1016/j.ymeth.2023.10.003","url":null,"abstract":"<div><p>Platelets are small circulating fragments of cells that play important roles in thrombosis, haemostasis, immune response, inflammation and cancer growth. Although anucleate, they contain a rich RNA repertoire which offers an opportunity to characterise changes in platelet gene expression in health and disease. Whilst this can be achieved with conventional RNA sequencing, a large input of high-quality RNA, and hence blood volume, is required (unless a pre-amplification step is added), along with specialist bioinformatic skills for data analysis and interpretation. We have developed a transcriptomics next-generation sequencing-based approach that overcomes these limitations. Termed PlateletSeq, this method requires very low levels of RNA input and does not require specialist bioinformatic analytical skills. Here we describe the methodology, from sample collection to processing and data analysis. Specifically, blood samples can be stored for up to 8 days at 4 °C prior to analysis. Platelets are isolated using multi-step centrifugation and a purity of ≤ 1 leucocyte per 0.26x10⁶ platelets is optimal for gene expression analysis. We have applied PlateletSeq to normal adult blood samples and show there are no age-associated variations and only minor gender-associated differences. In contrast, platelets from patients with myeloproliferative neoplasms show differences in platelet transcript profiles from normal and between disease subtypes. This illustrates the potential applicability of PlateletSeq for biomarker discovery and studying platelet biology in patient samples. It also opens avenues for assessing platelet quality in other fields such as transfusion research.</p></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"219 ","pages":"Pages 139-149"},"PeriodicalIF":4.8,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046202323001676/pdfft?md5=19652968fef800dc1386d77a86473fdd&pid=1-s2.0-S1046202323001676-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41181603","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pyrene-based fluorescent chemosensor for rapid detection of water and its applications","authors":"Pragyan P. Dash ,&nbsp;P. Mohanty ,&nbsp;S. Behera ,&nbsp;R. Behura ,&nbsp;Bibhuti B. Palai ,&nbsp;Bhaskar Nath ,&nbsp;Suban K. Sahoo ,&nbsp;Bigyan R. Jali","doi":"10.1016/j.ymeth.2023.10.006","DOIUrl":"10.1016/j.ymeth.2023.10.006","url":null,"abstract":"<div><p>This manuscript introduces a pyrene-based Schiff base <strong>L</strong> by reacting pyrenecarboxaldehyde with 2-aminothiazole in equimolar ratio. The ligand <strong>L</strong> was characterized by various spectral data and single crystal. The water sensing ability of <strong>L</strong><span> was examined in different organic solvents. The weakly emissive </span><strong>L</strong> in DMSO showed a fluorescence enhancement upon the addition of water. The water-induced fluorescence enhancement of <strong>L</strong><span> was occurred due to the combined effect of aggregation-induced emission (AIE) phenomenon and suppression of photo-induced electron transfer (PET) process. Using </span><strong>L</strong>, the water in DMSO can be detected down to 0.50 wt% with a quantification limit of 1.52 wt%. The analytical novelty of the developed sensor <strong>L</strong> was validated by detecting moisture in a variety of raw food products.</p></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"219 ","pages":"Pages 127-138"},"PeriodicalIF":4.8,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41187309","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}

{"title":"From correlation to causation using directed topological overlap matrix: Applications in genomics","authors":"Borzou Alipourfard ,&nbsp;Jean Gao","doi":"10.1016/j.ymeth.2023.09.005","DOIUrl":"10.1016/j.ymeth.2023.09.005","url":null,"abstract":"<div><p><span>Most causal discovery tools assume the local causal Markov condition. However, the theoretical assumptions that underlie the local causal Markov condition are often not met in practice. This is especially marked in genomics, where the unwanted presence of measurement errors, averaging effects, and feedback loops significantly undermine the legitimacy of the local causal Markov condition. Furthermore, these causal discovery algorithms require very large samples, orders above what is often available. In this paper, relaxing the local causal Markov condition and using Reichenbach's common cause principle instead, we present a more flexible approach to causal discovery, the directed topological overlap matrix (DTOM). DTOM is robust w.r.t. the presence of measurement errors, averaging effects, feedback loops, and is significantly more sample efficient. We study the utility of DTOM for discovering causal relations in biological data using three real gene expression data-sets. We first examine if DTOM can help distinguish the Myostatin mutation in the Piedmontese cattle by contrasting the muscle </span>transcriptomes<span> of the Piedmontese and Wagyu crosses: the Myostatin mutation is the cause of the double-muscling the Piedmontese cattle are famous for. We then consider a large-scale gene deletion study in yeast. We show that DTOM allows us to distinguish the deleted gene in a sample knowing only the set of differentially expressed genes in that sample. We then examine the progression of Alzheimer's disease (AD) under the lens of DTOM. The genes implicated as having a causal role in the progression of AD by our DTOM analysis were significantly enriched in cellular components that had been repeatedly implicated in the progression of AD.</span></p></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"219 ","pages":"Pages 58-67"},"PeriodicalIF":4.8,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41105243","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}

{"title":"Analysis of non-canonical three- and four-way DNA junctions","authors":"Bríonna McGorman ,&nbsp;Simon Poole ,&nbsp;Miguel Vázquez López ,&nbsp;Andrew Kellett","doi":"10.1016/j.ymeth.2023.09.002","DOIUrl":"10.1016/j.ymeth.2023.09.002","url":null,"abstract":"<div><p>The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs) and four-way Holliday junctions (HJs), offer an intriguing target for developmental therapeutics as both 3WJs and HJs are involved in DNA replication and repair processes. However, there are a limited number of assays available for the analysis of junction DNA binding. Here, we describe the design and execution of multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) and microscale thermophoresis (MST) assays that enable evaluation of junction-binding compounds. Two well characterised junction-binding compounds—a C6 linked bis-acridine ligand and an iron(II)-bound peptide helicate, which recognise HJs and 3WJs, respectively—were employed as probes for both MST and PAGE experiments. The multiplex PAGE assay expands beyond previously reported fluorescent PAGE as it uses four individual fluorophores that can be combined to visualise single-strands, pseudo-duplexes, and junction DNA present during 3WJ and HJ formation. The use of MST to identify the binding affinity of junction binding agents is, to our knowledge, first reported example of this technique. The combined use of PAGE and MST provides complementary results for the visualisation of 3WJ and HJ formation and the direct binding affinity (K_d and EC₅₀) of these agents. These assays can be used to aid the discovery and design of new therapeutics targeting non-canonical nucleic acid structures.</p></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"219 ","pages":"Pages 30-38"},"PeriodicalIF":4.8,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046202323001457/pdfft?md5=970ceed92843b153b068d80d084b3328&pid=1-s2.0-S1046202323001457-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10569966","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Does protein pretrained language model facilitate the prediction of protein–ligand interaction?","authors":"Weihong Zhang ,&nbsp;Fan Hu ,&nbsp;Wang Li ,&nbsp;Peng Yin","doi":"10.1016/j.ymeth.2023.08.016","DOIUrl":"10.1016/j.ymeth.2023.08.016","url":null,"abstract":"<div><p>Protein-ligand interaction (PLI) is a critical step for drug discovery. Recently, protein pretrained language models (PLMs) have showcased exceptional performance across a wide range of protein-related tasks. However, a significant heterogeneity exists between the PLM and PLI tasks, leading to a degree of uncertainty. In this study, we propose a method that quantitatively assesses the significance of protein PLMs in PLI prediction. Specifically, we analyze the performance of three widely-used protein PLMs (TAPE, ESM-1b, and ProtTrans) on three PLI tasks (PDBbind, Kinase, and DUD-E). The model with pre-training consistently achieves improved performance and decreased time cost, demonstrating that enhance both the accuracy and efficiency of PLI prediction. By quantitatively assessing the transferability, the optimal PLM for each PLI task is identified without the need for costly transfer experiments. Additionally, we examine the contributions of PLMs on the distribution of feature space, highlighting the improved discriminability after pre-training. Our findings provide insights into the mechanisms underlying PLMs in PLI prediction and pave the way for the design of more interpretable and accurate PLMs in the future. Code and data are freely available at <span>https://github.com/brian-zZZ/PLM-PLI</span><svg><path></path></svg>.</p></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"219 ","pages":"Pages 8-15"},"PeriodicalIF":4.8,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10242928","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}

{"title":"Exploring the artificial intelligence and machine learning models in the context of drug design difficulties and future potential for the pharmaceutical sectors","authors":"Periyasamy Natarajan Shiammala ,&nbsp;Navaneetha Krishna Bose Duraimutharasan ,&nbsp;Baskaralingam Vaseeharan ,&nbsp;Abdulaziz S. Alothaim ,&nbsp;Esam S. Al-Malki ,&nbsp;Babu Snekaa ,&nbsp;Sher Zaman Safi ,&nbsp;Sanjeev Kumar Singh ,&nbsp;Devadasan Velmurugan ,&nbsp;Chandrabose Selvaraj","doi":"10.1016/j.ymeth.2023.09.010","DOIUrl":"10.1016/j.ymeth.2023.09.010","url":null,"abstract":"<div><p>Artificial intelligence (AI), particularly deep learning as a subcategory of AI, provides opportunities to accelerate and improve the process of discovering and developing new drugs. The use of AI in drug discovery is still in its early stages, but it has the potential to revolutionize the way new drugs are discovered and developed. As AI technology continues to evolve, it is likely that AI will play an even greater role in the future of drug discovery. AI is used to identify new drug targets, design new molecules, and predict the efficacy and safety of potential drugs. The inclusion of AI in drug discovery can screen millions of compounds in a matter of hours, identifying potential drug candidates that would have taken years to find using traditional methods. AI is highly utilized in the pharmaceutical industry by optimizing processes, reducing waste, and ensuring quality control. This review covers much-needed topics, including the different types of machine-learning techniques, their applications in drug discovery, and the challenges and limitations of using machine learning in this field. The state-of-the-art of AI-assisted pharmaceutical discovery is described, covering applications in structure and ligand-based virtual screening, <em>de novo</em><span> drug creation, prediction of physicochemical and pharmacokinetic properties, drug repurposing, and related topics. Finally, many obstacles and limits of present approaches are outlined, with an eye on potential future avenues for AI-assisted drug discovery and design.</span></p></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"219 ","pages":"Pages 82-94"},"PeriodicalIF":4.8,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41176857","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}