Pub Date : 2025-10-30DOI: 10.1016/j.ymeth.2025.10.008
Donghyeok Gang, Yeonju Song, Yeonjin Ko
Approximately 80% of drugs developed to date are small molecule compounds. While these compounds can effectively inhibit intracellular targets by crossing cell membranes, their efficacy often depends on stringent conditions, such as the presence of a deep hydrophobic pocket for strong binding. Biologics—including peptides, antibodies, and genetic materials—have fewer binding requirements but cannot penetrate cell membranes, limiting their activity to extracellular targets. Notably, the number of intracellular protein and nucleic acid targets is more than four times that of extracellular targets. Given their potential to treat fundamental disease mechanisms, the intracellular delivery of biologics is of critical importance. In this review, we discuss the generation and application of membrane-based carriers, including cell-derived vesicles and artificial membrane-based carriers, with examples categorized by modality to enhance the therapeutic utility of biologics.
{"title":"Membrane-mediated strategies for efficient intracellular delivery of biologics","authors":"Donghyeok Gang, Yeonju Song, Yeonjin Ko","doi":"10.1016/j.ymeth.2025.10.008","DOIUrl":"10.1016/j.ymeth.2025.10.008","url":null,"abstract":"<div><div>Approximately 80% of drugs developed to date are small molecule compounds. While these compounds can effectively inhibit intracellular targets by crossing cell membranes, their efficacy often depends on stringent conditions, such as the presence of a deep hydrophobic pocket for strong binding. Biologics—including peptides, antibodies, and genetic materials—have fewer binding requirements but cannot penetrate cell membranes, limiting their activity to extracellular targets. Notably, the number of intracellular protein and nucleic acid targets is more than four times that of extracellular targets. Given their potential to treat fundamental disease mechanisms, the intracellular delivery of biologics is of critical importance. In this review, we discuss the generation and application of membrane-based carriers, including cell-derived vesicles and artificial membrane-based carriers, with examples categorized by modality to enhance the therapeutic utility of biologics.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"245 ","pages":"Pages 13-24"},"PeriodicalIF":4.3,"publicationDate":"2025-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145420758","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 : 2025-10-28DOI: 10.1016/j.ymeth.2025.10.009
Ahmad Itani , Marta Velaz Martín , Laura Meisch , Phillip Lemke , Tim Scharnweber , Islam M. Khattab , Kersten S. Rabe , Christof M. Niemeyer
Glyphosate, the active ingredient in many broad-spectrum herbicides, is extensively used in agriculture but has come under increasing scrutiny due to its potential impacts on non-target microbial communities. To investigate these effects within a controlled yet ecologically relevant framework, Winogradsky columns, self-contained sediment-based ecosystems, were employed as a model system. A novel, non-destructive sampling approach was introduced using macroporous elastomeric silicone foam (MESIF) integrated in stainless-steel frames to enable spatiotemporal monitoring of benthic microbial communities. These MESIF-loaded frames were vertically embedded in columns filled with lake sediment and subjected to varying experimental conditions, including light exposure and glyphosate treatment. Microbial colonization of the MESIF was assessed via amplicon sequencing at defined time points. Glyphosate-treated columns exhibited delayed microbial stratification and diminished development of characteristic pigmentation associated with functional groups such as iron-oxidizing and sulfate-reducing bacteria. Although within-column alpha diversity remained relatively constant, glyphosate exposure led to distinct shifts in community composition, including an increased abundance of taxa potentially involved in glyphosate degradation. These findings demonstrate the effectiveness of combining Winogradsky columns with MESIF-based sampling for studying environmental stressors and underscore glyphosate’s influence on microbial succession and functional diversity in sediment ecosystems.
{"title":"Materials-Based spatiotemporal analysis of microbial responses to glyphosate in Winogradsky columns","authors":"Ahmad Itani , Marta Velaz Martín , Laura Meisch , Phillip Lemke , Tim Scharnweber , Islam M. Khattab , Kersten S. Rabe , Christof M. Niemeyer","doi":"10.1016/j.ymeth.2025.10.009","DOIUrl":"10.1016/j.ymeth.2025.10.009","url":null,"abstract":"<div><div>Glyphosate, the active ingredient in many broad-spectrum herbicides, is extensively used in agriculture but has come under increasing scrutiny due to its potential impacts on non-target microbial communities. To investigate these effects within a controlled yet ecologically relevant framework, Winogradsky columns, self-contained sediment-based ecosystems, were employed as a model system. A novel, non-destructive sampling approach was introduced using macroporous elastomeric silicone foam (MESIF) integrated in stainless-steel frames to enable spatiotemporal monitoring of benthic microbial communities. These MESIF-loaded frames were vertically embedded in columns filled with lake sediment and subjected to varying experimental conditions, including light exposure and glyphosate treatment. Microbial colonization of the MESIF was assessed via amplicon sequencing at defined time points. Glyphosate-treated columns exhibited delayed microbial stratification and diminished development of characteristic pigmentation associated with functional groups such as iron-oxidizing and sulfate-reducing bacteria. Although within-column alpha diversity remained relatively constant, glyphosate exposure led to distinct shifts in community composition, including an increased abundance of taxa potentially involved in glyphosate degradation. These findings demonstrate the effectiveness of combining Winogradsky columns with MESIF-based sampling for studying environmental stressors and underscore glyphosate’s influence on microbial succession and functional diversity in sediment ecosystems.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"245 ","pages":"Pages 1-12"},"PeriodicalIF":4.3,"publicationDate":"2025-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145407820","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 : 2025-10-16DOI: 10.1016/j.ymeth.2025.10.006
Andreea Iuliana Ftodiev , Georgiana Necula Petrareanu , Mihaela Puiu , Gheorghe Proteasa , Cristian V.A. Munteanu , Roberta Maria Banciu , Ruchika Chauhan , Diana Visinescu , Cristina Purcarea , Pablo Fanjul-Bolado , David Ibañez , Ronen Fogel , Janice Limson , Monica Potara , Anca Florina Bonciu , Simion Astilean , Camelia Bala , Alina Vasilescu
The selective detection of dithiocarbamate fungicides in food and agricultural products presents significant analytical challenges. While Surface-enhanced Raman spectroscopy (SERS) has been extensively investigated to address this, detection systems based on enzymatic inhibition remain underexplored. Using thiram as a model dithiocarbamate, the present work explores the potential application of a cold-active aldehyde dehydrogenase from Flavobacterium PL002 for the development of specific, inhibition-based analytical methods. A molecular modelling and docking study confirmed that thiram fits into the binding pocket of the enzyme. An irreversible inhibition mechanism was inferred for thiram based on enzymatic kinetics studies. The mechanism was supported by SERS, mass spectrometry measurements and tests with reducing agents. A simple assay for the detection of the fungicide was developed and compared to a SERS-based procedure. The advantages and the practical limitations of the two methods were revealed by studying the detection of thiram from the surface of fungicide-spiked tomatoes. By coupling enzymatic inhibition with SERS, the selectivity for the detection of individual fungicides can be increased, as illustrated by comparing thiram with ziram, a structurally related compound. The study serves as basis for the development of analytical methods for the selective detection of thiram.
{"title":"Comprehensive analysis of the inhibition of aldehyde dehydrogenase from Flavobacterium PL002 and its coupling with SERS as a path for the selective detection of thiram","authors":"Andreea Iuliana Ftodiev , Georgiana Necula Petrareanu , Mihaela Puiu , Gheorghe Proteasa , Cristian V.A. Munteanu , Roberta Maria Banciu , Ruchika Chauhan , Diana Visinescu , Cristina Purcarea , Pablo Fanjul-Bolado , David Ibañez , Ronen Fogel , Janice Limson , Monica Potara , Anca Florina Bonciu , Simion Astilean , Camelia Bala , Alina Vasilescu","doi":"10.1016/j.ymeth.2025.10.006","DOIUrl":"10.1016/j.ymeth.2025.10.006","url":null,"abstract":"<div><div>The selective detection of dithiocarbamate fungicides in food and agricultural products presents significant analytical challenges. While Surface-enhanced Raman spectroscopy (SERS) has been extensively investigated to address this, detection systems based on enzymatic inhibition remain underexplored. Using thiram as a model dithiocarbamate, the present work explores the potential application of a cold-active aldehyde dehydrogenase from <em>Flavobacterium PL002</em> for the development of specific, inhibition-based analytical methods. A molecular modelling and docking study confirmed that thiram fits into the binding pocket of the enzyme. An irreversible inhibition mechanism was inferred for thiram based on enzymatic kinetics studies. The mechanism was supported by SERS, mass spectrometry measurements and tests with reducing agents. A simple assay for the detection of the fungicide was developed and compared to a SERS-based procedure. The advantages and the practical limitations of the two methods were revealed by studying the detection of thiram from the surface of fungicide-spiked tomatoes. By coupling enzymatic inhibition with SERS, the selectivity for the detection of individual fungicides can be increased, as illustrated by comparing thiram with ziram, a structurally related compound. The study serves as basis for the development of analytical methods for the selective detection of thiram.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"245 ","pages":"Pages 83-98"},"PeriodicalIF":4.3,"publicationDate":"2025-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145317960","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 : 2025-10-16DOI: 10.1016/j.ymeth.2025.10.007
Alicia del Prado , María I. Martínez-Jiménez , Luis Blanco, Miguel de Vega
As shown here, isothermal and primer-less amplification of specific padlock probes allows direct detection of SARS-CoV2 RNA without needing for a reverse transcription step. This simplified method of Hyperbranched Rolling Circle Amplification (HRCA) only requires three enzymes: SplintR (to ligate a specific padlock probe to its circular form, only when viral RNA is present), the unique DNA primase TthPrimPol (to generate de novo DNA primers), and an engineered and thermostabilized variant of bacteriophage phi29 DNA polymerase (phi29 DNApol), named Qx5. Qx5 is significantly more efficient than wild-type phi29 DNApol and shows an unexpectedly strong 3′-5′ exoribonucleolytic activity capable of trimming the 3′ polyA tail of the RNA target, thus enabling it as a primer for Qx5 to start Rolling Circle Amplification (RCA) of a nearby circularized padlock. The RCA step, that yields a long ssDNA concatemeric product (RCA product, RCP) is coupled to a second isothermal amplification step, assisted by TthPrimPol by synthesizing DNA primers on the RCP, that triggers exponential HRCA of the padlock sequence, catalyzed by Qx5. As a proof of concept, the application of this method for detection of SARS-CoV2 RNA rendered a significant amount of DNA in just 2 h at 37 °C, that can be easily evidenced by colorimetry, or even quantitated with a pocket fluorometer, and served for a quick diagnosis of SARS-CoV2 RNA infected versus non-infected samples.
如图所示，特定挂锁探针的等温和无引物扩增允许直接检测SARS-CoV2 RNA，而无需逆转录步骤。这种简化的Hyperbranched Rolling Circle Amplification （HRCA）方法只需要三种酶：SplintR（仅当病毒RNA存在时，将特定的锁探针连接到其圆形形式），独特的DNA引物酶thprimpol（生成新的DNA引物），以及噬菌体phi29 DNA聚合酶（phi29 DNApol）的工程和热稳定变体，命名为Qx5。Qx5明显比野生型phi29 DNApol更有效，并且显示出意想不到的强3‘-5’外核糖核分解活性，能够修剪RNA靶标的3' polyA尾部，从而使其成为Qx5启动附近环状锁的滚动圈扩增（RCA）的引物。RCA步骤，产生一个长ssDNA串联产物（RCA产物，RCP），与第二个等温扩增步骤耦合，由thprimpol辅助，通过在RCP上合成DNA引物，触发挂锁序列的指数HRCA，由Qx5催化。作为概念验证，将该方法应用于检测SARS-CoV2 RNA，在37 °C下仅需2 h即可获得大量DNA，这可以很容易地通过比色法证明，甚至可以用袖珍荧光仪定量，并用于快速诊断SARS-CoV2 RNA感染与非感染样品。
{"title":"Novel primer-less amplification method for detection of RNA molecules","authors":"Alicia del Prado , María I. Martínez-Jiménez , Luis Blanco, Miguel de Vega","doi":"10.1016/j.ymeth.2025.10.007","DOIUrl":"10.1016/j.ymeth.2025.10.007","url":null,"abstract":"<div><div>As shown here, isothermal and primer-less amplification of specific padlock probes allows direct detection of SARS-CoV2 RNA without needing for a reverse transcription step. This simplified method of Hyperbranched Rolling Circle Amplification (HRCA) only requires three enzymes: SplintR (to ligate a specific padlock probe to its circular form, only when viral RNA is present), the unique DNA primase <em>Tth</em>PrimPol (to generate <em>de novo</em> DNA primers), and an engineered and thermostabilized variant of bacteriophage phi29 DNA polymerase (phi29 DNApol), named Qx5. Qx5 is significantly more efficient than wild-type phi29 DNApol and shows an unexpectedly strong 3′-5′ exoribonucleolytic activity capable of trimming the 3′ polyA tail of the RNA target, thus enabling it as a primer for Qx5 to start Rolling Circle Amplification (RCA) of a nearby circularized padlock. The RCA step, that yields a long ssDNA concatemeric product (RCA product, RCP) is coupled to a second isothermal amplification step, assisted by <em>Tth</em>PrimPol by synthesizing DNA primers on the RCP, that triggers exponential HRCA of the padlock sequence, catalyzed by Qx5. As a proof of concept, the application of this method for detection of SARS-CoV2 RNA rendered a significant amount of DNA in just 2 h at 37 °C, that can be easily evidenced by colorimetry, or even quantitated with a pocket fluorometer, and served for a quick diagnosis of SARS-CoV2 RNA infected <em>versus</em> non-infected samples.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 227-239"},"PeriodicalIF":4.3,"publicationDate":"2025-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145318003","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 : 2025-10-14DOI: 10.1016/j.ymeth.2025.10.005
Xiaodong Zhang , Caixia Li , Yongzhen Wang , Yue Cao , Xiaona He , Fugang Xiao , Deguo Wang
Panax notoginseng, a cornerstone of traditional Chinese medicine, is frequently subject to adulteration in commercial markets, compromising its therapeutic efficacy and safety. This study introduces a novel application of Proofman-LMTIA technology to authenticate P. notoginseng with high precision and efficiency. By targeting unique sequence variations in the ITS2 rDNA region, we developed species-specific primers and probe to distinguish P. notoginseng from common adulterants. The method achieves a detection sensitivity of 10 pg/µL and identifies adulteration at levels as low as 1 % (v/v), validated across diverse commercial products, including powders and capsules. With a detection time of under 30 min and no reliance on specialized equipment, this approach offers a streamlined, cost-efficient solution for quality assurance in the herbal industry. Our results demonstrate 100 % accuracy in market sample testing, addressing critical challenges in P. notoginseng authentication and supporting regulatory compliance.
{"title":"Authentication of Panax notoginseng with high-efficiency Proofman-LMTIA technology","authors":"Xiaodong Zhang , Caixia Li , Yongzhen Wang , Yue Cao , Xiaona He , Fugang Xiao , Deguo Wang","doi":"10.1016/j.ymeth.2025.10.005","DOIUrl":"10.1016/j.ymeth.2025.10.005","url":null,"abstract":"<div><div><em>Panax notoginseng</em>, a cornerstone of traditional Chinese medicine, is frequently subject to adulteration in commercial markets, compromising its therapeutic efficacy and safety. This study introduces a novel application of Proofman-LMTIA technology to authenticate <em>P. notoginseng</em> with high precision and efficiency. By targeting unique sequence variations in the ITS2 rDNA region, we developed species-specific primers and probe to distinguish <em>P. notoginseng</em> from common adulterants. The method achieves a detection sensitivity of 10 pg/µL and identifies adulteration at levels as low as 1 % (v/v), validated across diverse commercial products, including powders and capsules. With a detection time of under 30 min and no reliance on specialized equipment, this approach offers a streamlined, cost-efficient solution for quality assurance in the herbal industry. Our results demonstrate 100 % accuracy in market sample testing, addressing critical challenges in <em>P</em>. <em>notoginseng</em> authentication and supporting regulatory compliance.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 219-226"},"PeriodicalIF":4.3,"publicationDate":"2025-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145306601","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 : 2025-10-13DOI: 10.1016/j.ymeth.2025.09.006
Xinfu Liu , Yirui Wu , Yuting Zhou
With the continuous advancement of medical enterprise, intelligent medical technologies supported by natural language processing and knowledge representation have made significant progress. However, with the continuous generation of vast amounts of medical data, the current methods still perform poorly in handling specialized medical data, particularly unlabeled medical diagnostic data. Inspired by the outstanding performance of large language models in various downstream expert tasks in recent years, this article leverages large language models to handle the massive unlabelled medical data, aiming to provide more accurate technical solutions for medical image classification tasks. Specifically, we propose a novel Cross-Modal Knowledge Representation framework (CMKR) to handle vast unlabeled medical data, which utilizes large language models to extract implicit knowledge from medical images, while also extracting explicit textual knowledge with the aid of knowledge graphs. To better utilize the associative information between medical images and textual records, we have designed a cross-modal alignment strategy that enhances knowledge representation capabilities both intra- and inter-modal. We conducted extensive experiments on public datasets, demonstrating that our method outperforms most mainstream approaches.
{"title":"Zero-shot medical image classification via large multimodal models and knowledge graphs-driven processing","authors":"Xinfu Liu , Yirui Wu , Yuting Zhou","doi":"10.1016/j.ymeth.2025.09.006","DOIUrl":"10.1016/j.ymeth.2025.09.006","url":null,"abstract":"<div><div>With the continuous advancement of medical enterprise, intelligent medical technologies supported by natural language processing and knowledge representation have made significant progress. However, with the continuous generation of vast amounts of medical data, the current methods still perform poorly in handling specialized medical data, particularly unlabeled medical diagnostic data. Inspired by the outstanding performance of large language models in various downstream expert tasks in recent years, this article leverages large language models to handle the massive unlabelled medical data, aiming to provide more accurate technical solutions for medical image classification tasks. Specifically, we propose a novel Cross-Modal Knowledge Representation framework (CMKR) to handle vast unlabeled medical data, which utilizes large language models to extract implicit knowledge from medical images, while also extracting explicit textual knowledge with the aid of knowledge graphs. To better utilize the associative information between medical images and textual records, we have designed a cross-modal alignment strategy that enhances knowledge representation capabilities both intra- and inter-modal. We conducted extensive experiments on public datasets, demonstrating that our method outperforms most mainstream approaches.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"245 ","pages":"Pages 25-34"},"PeriodicalIF":4.3,"publicationDate":"2025-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145297807","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 : 2025-10-12DOI: 10.1016/j.ymeth.2025.10.004
Kusum Kharga , Deepak Kumar , Lokender Kumar
Antimicrobial resistance (AMR) has emerged as a global crisis, responsible for millions of deaths annually. Traditionally used antimicrobial susceptibility testing (AST) methods, such as disk diffusion and broth dilution, suffer from limitations including prolonged incubation time, inconsistent results, and high instrumentation cost. To address these challenges, we present in this study our novel, cost-effective antimicrobial detection assay equipped with a highly sensitive quantification system, LabImageXpert. Our antimicrobial detection system leverages the ability of β-galactosidase to convert colorless X-gal to an intense insoluble blue colored product. Unlike traditional MIC assays, LabImageXpert enables image-based, mechanism-specific growth assessment. During our investigation, we tested compounds already known to possess antimicrobial properties against our antimicrobial assay and found that it could not only detect the antimicrobial activity but also reveal the mechanism of action behind their antimicrobial activity, i.e., bacteriostatic and bacteriolytic. Further, this system contains a high-resolution DSLR camera to image samples in a microtiter plate placed inside a special box. The captured images are processed using the open-source software. We performed an experiment exclusively to test its ability to quantify minute variations in bacterial growth over time. The results showed that it could detect minute deviations in average pixel intensity over time. LabImageXpert detected bacteriolytic activity of compounds within 3 h. Our findings suggest that the LabImageXpert provides a reproducible, scalable, and cost-effective alternative platform for antimicrobial research in teaching, research and clinical diagnostic labs.
{"title":"LabImageXpert: imaging platform for mechanism-resolved detection of antimicrobial activity via β-galactosidase assay","authors":"Kusum Kharga , Deepak Kumar , Lokender Kumar","doi":"10.1016/j.ymeth.2025.10.004","DOIUrl":"10.1016/j.ymeth.2025.10.004","url":null,"abstract":"<div><div>Antimicrobial resistance (AMR) has emerged as a global crisis, responsible for millions of deaths annually. Traditionally used antimicrobial susceptibility testing (AST) methods, such as disk diffusion and broth dilution, suffer from limitations including prolonged incubation time, inconsistent results, and high instrumentation cost. To address these challenges, we present in this study our novel, cost-effective antimicrobial detection assay equipped with a highly sensitive quantification system, LabImageXpert. Our antimicrobial detection system leverages the ability of β-galactosidase to convert colorless X-gal to an intense insoluble blue colored product. Unlike traditional MIC assays, LabImageXpert enables image-based, mechanism-specific growth assessment. During our investigation, we tested compounds already known to possess antimicrobial properties against our antimicrobial assay and found that it could not only detect the antimicrobial activity but also reveal the mechanism of action behind their antimicrobial activity, i.e., bacteriostatic and bacteriolytic. Further, this system contains a high-resolution DSLR camera to image samples in a microtiter plate placed inside a special box. The captured images are processed using the open-source software. We performed an experiment exclusively to test its ability to quantify minute variations in bacterial growth over time. The results showed that it could detect minute deviations in average pixel intensity over time. LabImageXpert detected bacteriolytic activity of compounds within 3 h. Our findings suggest that the LabImageXpert provides a reproducible, scalable, and cost-effective alternative platform for antimicrobial research in teaching, research and clinical diagnostic labs.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 182-194"},"PeriodicalIF":4.3,"publicationDate":"2025-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145290536","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 : 2025-10-11DOI: 10.1016/j.ymeth.2025.10.003
Ayoub Shiravandi , Ibrahim Zarkesh , Melina Ghasemian , Mohammad Kazemi Ashtiani , Gholamreza Bayazian , Alimohamad Asghari , Rafieh Alizadeh , Zohreh Bagher , Seyed Mohammad Davachi
Soft tissue engineering represents a transformative solution for regenerating damaged tissues, with the development of advanced hydrogels playing a crucial role in this process. Gelatin methacryloyl (GelMA) has emerged as a promising material due to its biocompatibility and ability to support cell growth; however, its mechanical limitations have driven the exploration of composite hydrogels, with thiolated graphene oxide (tGONS) standing out as a key additive. In this study, we present a novel strategy involving thiolated graphene oxide nanosheets (tGONS) as covalent crosslinkers within the GelMA hydrogel matrix, wherein thiol groups on tGONS react with GelMA’s acrylate groups, enhancing crosslinking and strengthening the hydrogel network. The results demonstrate that incorporation of tGONS at optimized concentrations (≤0.5 mg/mL) leads to a substantial increase in Young’s modulus and gel fraction, along with a marked enhancement in antioxidant activity, evidenced by up to 40 % DPPH and 60 % H2O2 scavenging in G1GTII formulations. Despite the increased stiffness, acceptable elongation and high cytocompatibility (>85 % cell viability) were maintained. Additionally, the composite hydrogels showed improved resistance to enzymatic degradation and reduced swelling, offering more precise control over physicochemical properties.These findings highlight the multifunctional advantages of GelMA–tGONS composite hydrogels, positioning them as adaptable and robust scaffolds suitable for applications in soft tissue engineering, including cartilage, dermal, and neural regeneration.
{"title":"Gelatin methacryloyl − thiolated graphene oxide composites for soft tissue engineering","authors":"Ayoub Shiravandi , Ibrahim Zarkesh , Melina Ghasemian , Mohammad Kazemi Ashtiani , Gholamreza Bayazian , Alimohamad Asghari , Rafieh Alizadeh , Zohreh Bagher , Seyed Mohammad Davachi","doi":"10.1016/j.ymeth.2025.10.003","DOIUrl":"10.1016/j.ymeth.2025.10.003","url":null,"abstract":"<div><div>Soft tissue engineering represents a transformative solution for regenerating damaged tissues, with the development of advanced hydrogels playing a crucial role in this process. Gelatin methacryloyl (GelMA) has emerged as a promising material due to its biocompatibility and ability to support cell growth; however, its mechanical limitations have driven the exploration of composite hydrogels, with thiolated graphene oxide (tGONS) standing out as a key additive. In this study, we present a novel strategy involving thiolated graphene oxide nanosheets (tGONS) as covalent crosslinkers within the GelMA hydrogel matrix, wherein thiol groups on tGONS react with GelMA’s acrylate groups, enhancing crosslinking and strengthening the hydrogel network. The results demonstrate that incorporation of tGONS at optimized concentrations (≤0.5 mg/mL) leads to a substantial increase in Young’s modulus and gel fraction, along with a marked enhancement in antioxidant activity, evidenced by up to 40 % DPPH and 60 % H<sub>2</sub>O<sub>2</sub> scavenging in G1GTII formulations. Despite the increased stiffness, acceptable elongation and high cytocompatibility (>85 % cell viability) were maintained. Additionally, the composite hydrogels showed improved resistance to enzymatic degradation and reduced swelling, offering more precise control over physicochemical properties.These findings highlight the multifunctional advantages of GelMA–tGONS composite hydrogels, positioning them as adaptable and robust scaffolds suitable for applications in soft tissue engineering, including cartilage, dermal, and neural regeneration.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 170-181"},"PeriodicalIF":4.3,"publicationDate":"2025-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145285367","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 : 2025-10-09DOI: 10.1016/j.ymeth.2025.10.002
Yogita Jethmalani , Matthew S. Sutton , Robin Carroll , Rachel Kazmierski , Kwang Low , Madeeha Mughal , Jimmie Bullock , Cecilia S. Lindestam Arlehamn , David M. Lewinsohn , Deborah A. Lewinsohn , Chelsea C. Lehman , Courtney Green , James Moshi , Allen Mueller , Patricia A. Darrah , Robert A. Seder , Bob C. Lin , Richard A. Koup , Leonid A. Serebryannyy , Mario Roederer
Immunospot assays are known for high sensitivity and low material requirement. ELISpot and FluoroSpot assays have been frequently used in immune cell monitoring and profiling, specifically with T-cells and B-cells. FluoroSpot enables multiplexing, similar to flow cytometry, but has the added benefit of requiring fewer cells, higher throughput at screening immunogens, and a faster assay readout. Immunospot assays are generally performed manually and are prone to operator errors in plate handling, leading to overlapping spots with low resolution and high variability. Here, we describe the development of a High-throughput Immune Cell FluoroSpot (HI-CeFSpot) assay that has been adapted on the Biomek i7 liquid handler with labware storage, in conjunction with an automated plate washer with stacker, and the IRIS 2 plate reader from Mabtech attached to the Orbitor robotic arm. To develop the HI-CeFSpot assay, we used immune cells from non-human primates (NHPs) and screened them against various stimuli to test the release of interferon gamma (IFN-γ). We tested parameters such as precision, robustness, reproducibility, and compared two different cell types across various cell densities. We found that the HI-CeFSpot assay had intra- and inter-plate precision of <10 %, and inter-assay precision of <15 %. The assay showed high reproducibility and was robust across multiple samples. The HI-CeFSpot assay described here is a platform solution that can be used in clinical trial endpoint testing for drug development, immune cell monitoring, testing the efficacy of immunotherapy, and in vaccine research with high-throughput, high precision, reproducibility, and multiplexing.
{"title":"HI-CeFSpot: High-throughput Immune Cell FluoroSpot assay","authors":"Yogita Jethmalani , Matthew S. Sutton , Robin Carroll , Rachel Kazmierski , Kwang Low , Madeeha Mughal , Jimmie Bullock , Cecilia S. Lindestam Arlehamn , David M. Lewinsohn , Deborah A. Lewinsohn , Chelsea C. Lehman , Courtney Green , James Moshi , Allen Mueller , Patricia A. Darrah , Robert A. Seder , Bob C. Lin , Richard A. Koup , Leonid A. Serebryannyy , Mario Roederer","doi":"10.1016/j.ymeth.2025.10.002","DOIUrl":"10.1016/j.ymeth.2025.10.002","url":null,"abstract":"<div><div>Immunospot assays are known for high sensitivity and low material requirement. ELISpot and FluoroSpot assays have been frequently used in immune cell monitoring and profiling, specifically with T-cells and B-cells. FluoroSpot enables multiplexing, similar to flow cytometry, but has the added benefit of requiring fewer cells, higher throughput at screening immunogens, and a faster assay readout. Immunospot assays are generally performed manually and are prone to operator errors in plate handling, leading to overlapping spots with low resolution and high variability. Here, we describe the development of a <u>H</u>igh-throughput <u>I</u>mmune <u>Ce</u>ll <u>F</u>luoro<u>Spot</u> (HI-CeFSpot) assay that has been adapted on the Biomek i7 liquid handler with labware storage, in conjunction with an automated plate washer with stacker, and the IRIS 2 plate reader from Mabtech attached to the Orbitor robotic arm. To develop the HI-CeFSpot assay, we used immune cells from non-human primates (NHPs) and screened them against various stimuli to test the release of interferon gamma (IFN-γ). We tested parameters such as precision, robustness, reproducibility, and compared two different cell types across various cell densities. We found that the HI-CeFSpot assay had intra- and inter-plate precision of <10 %, and inter-assay precision of <15 %. The assay showed high reproducibility and was robust across multiple samples. The HI-CeFSpot assay described here is a platform solution that can be used in clinical trial endpoint testing for drug development, immune cell monitoring, testing the efficacy of immunotherapy, and in vaccine research with high-throughput, high precision, reproducibility, and multiplexing.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 210-218"},"PeriodicalIF":4.3,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145257070","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 : 2025-10-03DOI: 10.1016/j.ymeth.2025.09.011
Dong-Gyu Jeon , Chung-Young Lee , Chang-Hee Cho , Gang Ho Lee , Yongmin Chang , Sung-Wook Nam
We report a comparative study of macroscopic and microscopic optical absorbance in hemagglutination (HA) assay. Red blood cells (RBCs) exhibit unique optical absorbance properties with characteristic peaks including Soret, Qv, and Qo. In addition, RBCs absorb light and appear as dark contrast in bright-field microscopy images, indicating an increase in local optical density (OD). By systematic analysis of macroscopic and microscopic OD measurements and UV–Visible (UV–Vis) spectroscopy, we developed a phenomenological model of RBC agglutination and non-agglutination. The antigen–antibody reaction in RBC agglutination behaves as a catastrophic event such that networking of RBC clumps is initiated at a critical RBC concentration. We analyzed the dependence of OD on RBC concentration. At the critical RBC concentration, OD values are dropped or saturated for RBC agglutination, on the other hand, ODs keep increasing as the increase of RBC concentration for RBC non-agglutination. By the analysis of UV–Vis spectroscopy for HA assay, we provide an optimal wavelength range as 480-520 nm, away from RBC characteristic absorption peaks. For further validation, we demonstrated the OD-based HA assay for the detection of H1N1 influenza A virus. Our investigation provides insights into how to utilize the physical properties of RBCs for novel HA assay platforms.
{"title":"Comparative analysis of macroscopic and microscopic optical absorbance in hemagglutination assay","authors":"Dong-Gyu Jeon , Chung-Young Lee , Chang-Hee Cho , Gang Ho Lee , Yongmin Chang , Sung-Wook Nam","doi":"10.1016/j.ymeth.2025.09.011","DOIUrl":"10.1016/j.ymeth.2025.09.011","url":null,"abstract":"<div><div>We report a comparative study of macroscopic and microscopic optical absorbance in hemagglutination (HA) assay. Red blood cells (RBCs) exhibit unique optical absorbance properties with characteristic peaks including Soret, Qv, and Qo. In addition, RBCs absorb light and appear as dark contrast in bright-field microscopy images, indicating an increase in local optical density (OD). By systematic analysis of macroscopic and microscopic OD measurements and UV–Visible (UV–Vis) spectroscopy, we developed a phenomenological model of RBC agglutination and non-agglutination. The antigen–antibody reaction in RBC agglutination behaves as a catastrophic event such that networking of RBC clumps is initiated at a critical RBC concentration. We analyzed the dependence of OD on RBC concentration. At the critical RBC concentration, OD values are dropped or saturated for RBC agglutination, on the other hand, ODs keep increasing as the increase of RBC concentration for RBC non-agglutination. By the analysis of UV–Vis spectroscopy for HA assay, we provide an optimal wavelength range as 480-520 nm, away from RBC characteristic absorption peaks. For further validation, we demonstrated the OD-based HA assay for the detection of H1N1 influenza A virus. Our investigation provides insights into how to utilize the physical properties of RBCs for novel HA assay platforms.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 195-209"},"PeriodicalIF":4.3,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145231083","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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