Biology Methods and Protocols最新文献

{"title":"Robust RNA secondary structure prediction with a mixture of deep learning and physics-based experts.","authors":"Xiangyun Qiu","doi":"10.1093/biomethods/bpae097","DOIUrl":"https://doi.org/10.1093/biomethods/bpae097","url":null,"abstract":"<p><p>A mixture-of-experts (MoE) approach has been developed to mitigate the poor out-of-distribution (OOD) generalization of deep learning (DL) models for single-sequence-based prediction of RNA secondary structure. The main idea behind this approach is to use DL models for in-distribution (ID) test sequences to leverage their superior ID performances, while relying on physics-based models for OOD sequences to ensure robust predictions. One key ingredient of the pipeline, named MoEFold2D, is automated ID/OOD detection via consensus analysis of an ensemble of DL model predictions without requiring access to training data during inference. Specifically, motivated by the clustered distribution of known RNA structures, a collection of distinct DL models is trained by iteratively leaving one cluster out. Each DL model hence serves as an expert on all but one cluster in the training data. Consequently, for an ID sequence, all but one DL model makes accurate predictions consistent with one another, while an OOD sequence yields highly inconsistent predictions among all DL models. Through consensus analysis of DL predictions, test sequences are categorized as ID or OOD. ID sequences are subsequently predicted by averaging the DL models in consensus, and OOD sequences are predicted using physics-based models. Instead of remediating generalization gaps with alternative approaches such as transfer learning and sequence alignment, MoEFold2D circumvents unpredictable ID-OOD gaps and combines the strengths of DL and physics-based models to achieve accurate ID and robust OOD predictions.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpae097"},"PeriodicalIF":2.5,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11729747/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142985008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Real time-PCR a diagnostic tool for reporting copy number variation and relative gene-expression changes in pediatric B-cell acute lymphoblastic leukemia-a pilot study.","authors":"Zoha Sadaqat, Smitha Joseph, Chandrika Verma, Jyothi Muni Reddy, Anand Prakash, Tinku Thomas, Vandana Bharadwaj, Neha Vyas","doi":"10.1093/biomethods/bpae098","DOIUrl":"10.1093/biomethods/bpae098","url":null,"abstract":"<p><p>Real time-polymerase chain reaction (RT-PCR) is used routinely in clinical practice as a cost-effective method for molecular diagnostics. Research in pediatric B-cell Acute Lymphoblastic Leukemia (ped B-ALL) suggests that apart from cytogenetics and clinical features, there is a need to include Copy number variation (CNV) in select genes at diagnosis, for upfront stratification of patients. Using ped B-ALL as a model, we have developed a RT-PCR-based iterative probability scoring method for reporting CNVs, and relative gene-expression changes. Our work highlights that once genes of interest and hotspots of CNVs are identified in discovery phase, our proposed method can be used as a cost-effective and user-friendly diagnostic tool for the identification of changes at genomic or transcriptomic level. It has the potential to be incorporated in routine diagnostics in resource constrained settings and be tailored for different diseases as per need.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpae098"},"PeriodicalIF":2.5,"publicationDate":"2024-12-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717350/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An efficient injection protocol for <i>Drosophila</i> larvae.","authors":"Sattar Soltani, Nhan Huynh, Kirst King-Jones","doi":"10.1093/biomethods/bpae093","DOIUrl":"10.1093/biomethods/bpae093","url":null,"abstract":"<p><p>Intravenous injection provides a direct, rapid, and efficient route for delivering drugs or other substances, particularly for compounds with poor intestinal absorption or molecules (e.g. proteins) that are prone to structural changes and degradation within the digestive system. While <i>Drosophila</i> larvae represent a well-established genetic model for studying developmental and physiological pathways, as well as human diseases, their use in analyzing the molecular effects of substance exposure remains limited. In this study, we present a highly efficient injection method for <i>Drosophila</i> first- and second-instar larvae. Despite causing a slight developmental delay, this method achieves a high survival rate and offers a quick, easily adjustable protocol. The process requires 3-5 h to inject 150-300 larvae, depending on the microcapillary needle, microinjection system, and the compound being administered. As proof of concept, we compared the effects of injecting ferritin protein into <i>Fer1HCH⁰⁰⁴⁵¹</i> mutant first instar larvae with those of dietary ferritin administration. Our results show that ferritin injection rescues <i>Fer1HCH</i> mutants, a result that cannot be achieved through dietary delivery. This approach is particularly valuable for the delivery of complex compounds in cases where oral administration is impaired or limited by the digestive system.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae093"},"PeriodicalIF":2.5,"publicationDate":"2024-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11661979/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142878157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A cognitive and sensory approach based on workshops using the zebrafish model promotes the discovery of life sciences in the classroom.","authors":"Laure M Bourcier, Patrick J Babin","doi":"10.1093/biomethods/bpae092","DOIUrl":"10.1093/biomethods/bpae092","url":null,"abstract":"<p><p>The main objective of the ZebraCool programme was to create a positive attitude and curiosity towards science by bringing experimental activities within schools using an introductory cognitive and sensory approach. This innovative programme was offered at all levels of primary and secondary education including vocational high schools. Thematic workshops can be carried out on various themes such as comparative anatomy and embryology, molecular biology and evolution, or toxicology and endocrine disruptors. They were on an ad hoc basis or as part of an annual school project using zebrafish as a model. This animal was a very attractive entry point for the educator to motivate students to appreciate biology, in particular in the field of molecular biology and evolution. For each practical workshop, the student was an actor in his/her learning, which was intended to arouse the curiosity and desire to understand and learn. The programme was based on close collaboration between class teachers and programme educators to adapt workshops' content to the school curriculum. Students conducted their own experiments, formulated and tested hypotheses, learned laboratory techniques, collected, and analysed data. ZebraCool scientific activities fell within a conceptual framework of evolutionary biology through which participants perceived their own inner fish through the comparison of biological processes between humans and zebrafish.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae092"},"PeriodicalIF":2.5,"publicationDate":"2024-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11658831/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142865724","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protocol for obtaining doubled haploids in isolated microspore culture <i>in vitro</i> for poorly responsive genotypes of brassicaceae family.","authors":"Elena V Kozar, Elena A Domblides","doi":"10.1093/biomethods/bpae091","DOIUrl":"10.1093/biomethods/bpae091","url":null,"abstract":"<p><p>In this protocol for obtaining doubled haploids plants (DH), we propose a new method for microspore isolation. This method is useful for genotypes of the Brassicaceae family with low responsiveness to DH technology. For such crops, it allows increasing the embryo yield several times and sometimes obtaining embryos for the first time. This method of microspore isolation reduces the mechanical impact on the bud tissue, which minimizes somatic cell destruction and reduces to get it into the preparation through the filter, thus increasing its purity. The new isolation method also increases the relative concentration of embryogenic microspores in the preparation. This is possible because the anther tissues are not destroyed during the isolation process. Therefore, the anther retains its structure and microspores of early and late stages are trapped by the anther tissue, thus the anther acts as a sieve. Late stages are trapped because of their larger size, while early stages are trapped because they are even more tightly bound to the anther tissue. Together, these factors increase the efficiency of the technology for DH production <i>in vitro</i> microspore culture. This protocol article provides a detailed experimental protocol to the method presented in the experimental article (E.V. Kozar, E.G. Kozar, E.A. Domblides. Effect of the Method of Microspore Isolation on the Efficiency of Isolated Microspore Culture In Vitro for Brassicaceae Family. Horticulturae. 2022. Vol. 8, No. 10. P. 864. DOI 10.3390/horticulturae8100864) but does not repeat all the results documenting the efficacy of the actual method.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae091"},"PeriodicalIF":2.5,"publicationDate":"2024-12-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11668254/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142886176","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a large-scale rapid LAMP diagnostic testing platform for pandemic preparedness and outbreak response.","authors":"Rinie van Beuningen, Kin Ki Jim, Maikel Boot, Michel Ossendrijver, Bart J F Keijser, Jeroen H B van de Bovenkamp, Willem J G Melchers, Tim Kievits","doi":"10.1093/biomethods/bpae090","DOIUrl":"10.1093/biomethods/bpae090","url":null,"abstract":"<p><p>The coronavirus disease 2019 (COVID-19) pandemic underscored the necessity for rapid and efficient diagnostic testing to mitigate outbreaks and control disease transmission. While real-time reverse transcriptase quantitative PCR (RT-qPCR) has been the gold standard due to its high sensitivity and specificity, its logistical complexities and extended turnaround times highlighted the need for alternative molecular methods and non-standard equipment and consumables not subject to supply chain pressure. Loop-mediated isothermal amplification (LAMP) offers several advantages over RT-qPCR, including faster processing time, assay flexibility and cost-effectiveness. During the pandemic, LAMP was successfully demonstrated as a viable alternative to RT-qPCR for SARS-Related Coronavirus 2 detection. However, due to a 100 to 1,000-fold increase in testing volumes, there was an imminent need for automating and scaling up existing LAMP testing workflows leveraging a robotic infrastructure, while retaining analytical performance and cost-effectiveness. In 2020, the Foundation TOMi started the \"TOMi corona initiative\" to develop and validate a high-throughput, end-to-end, automated, scalable single-step RNA purification, and LAMP-based COVID-19 testing system called SMART-LAMP (Scalable Molecular Automation for Rapid Testing using LAMP) that can process up to 40,000 samples per day using existing laboratory equipment infrastructure with sensitivity comparable to RT-qPCR. This system provides a rapid and scalable diagnostic solution for future pandemics, capable of processing over 40,000 samples per day. In addition, the system is designed to minimize consumable costs and reduces the overall use of plastics to align with increasingly strict sustainability goals that will be imposed over the coming years. Importantly, this system and public-private partnerships in the TOMi corona initiative has the potential to serve as a baseline to enhance pandemic preparedness and response capabilities.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae090"},"PeriodicalIF":2.5,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11634539/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142814397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"iHDSel software: The price equation and the population stability index to detect genomic patterns compatible with selective sweeps. An example with SARS-CoV-2.","authors":"Antonio Carvajal-Rodríguez","doi":"10.1093/biomethods/bpae089","DOIUrl":"10.1093/biomethods/bpae089","url":null,"abstract":"<p><p>A large number of methods have been developed and continue to evolve for detecting the signatures of selective sweeps in genomes. Significant advances have been made, including the combination of different statistical strategies and the incorporation of artificial intelligence (machine learning) methods. Despite these advances, several common problems persist, such as the unknown null distribution of the statistics used, necessitating simulations and resampling to assign significance to the statistics. Additionally, it is not always clear how deviations from the specific assumptions of each method might affect the results. In this work, allelic classes of haplotypes are used along with the informational interpretation of the Price equation to design a statistic with a known distribution that can detect genomic patterns caused by selective sweeps. The statistic consists of Jeffreys divergence, also known as the population stability index, applied to the distribution of allelic classes of haplotypes in two samples. Results with simulated data show optimal performance of the statistic in detecting divergent selection. Analysis of real severe acute respiratory syndrome coronavirus 2 genome data also shows that some of the sites playing key roles in the virus's fitness and immune escape capability are detected by the method. The new statistic, called <i>J_HAC</i> , is incorporated into the iHDSel (informed HacDivSel) software available at https://acraaj.webs.uvigo.es/iHDSel.html.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae089"},"PeriodicalIF":2.5,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11646571/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142830126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction to: Heterozygous <i>KCNH2</i> variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology.","authors":"","doi":"10.1093/biomethods/bpae085","DOIUrl":"https://doi.org/10.1093/biomethods/bpae085","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.1093/biomethods/bpab003.].</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae085"},"PeriodicalIF":2.5,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11631474/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142808177","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deep learning and transfer learning for brain tumor detection and classification.","authors":"Faris Rustom, Ezekiel Moroze, Pedram Parva, Haluk Ogmen, Arash Yazdanbakhsh","doi":"10.1093/biomethods/bpae080","DOIUrl":"10.1093/biomethods/bpae080","url":null,"abstract":"<p><p>Convolutional neural networks (CNNs) are powerful tools that can be trained on image classification tasks and share many structural and functional similarities with biological visual systems and mechanisms of learning. In addition to serving as a model of biological systems, CNNs possess the convenient feature of transfer learning where a network trained on one task may be repurposed for training on another, potentially unrelated, task. In this retrospective study of public domain MRI data, we investigate the ability of neural network models to be trained on brain cancer imaging data while introducing a unique camouflage animal detection transfer learning step as a means of enhancing the networks' tumor detection ability. Training on glioma and normal brain MRI data, post-contrast T1-weighted and T2-weighted, we demonstrate the potential success of this training strategy for improving neural network classification accuracy. Qualitative metrics such as feature space and DeepDreamImage analysis of the internal states of trained models were also employed, which showed improved generalization ability by the models following camouflage animal transfer learning. Image saliency maps further this investigation by allowing us to visualize the most important image regions from a network's perspective while learning. Such methods demonstrate that the networks not only 'look' at the tumor itself when deciding, but also at the impact on the surrounding tissue in terms of compressions and midline shifts. These results suggest an approach to brain tumor MRIs that is comparable to that of trained radiologists while also exhibiting a high sensitivity to subtle structural changes resulting from the presence of a tumor.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae080"},"PeriodicalIF":2.5,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11631523/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142808179","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Practical application of PMA-qPCR assay for determination of viable cells of inter-species biofilm of <i>Candida albicans-Staphylococcus aureus</i>.","authors":"Samuel Kendra, Jarmila Czucz Varga, Barbora Gaálová-Radochová, Helena Bujdáková","doi":"10.1093/biomethods/bpae081","DOIUrl":"10.1093/biomethods/bpae081","url":null,"abstract":"<p><p>Determining the number of viable cells by calculating colony-forming units is time-consuming. The evaluation of mixed biofilms consisting of different species is particularly problematic. Therefore, the aim of this study was to optimize a molecular method-propidium monoazide quantitative polymerase chain reaction (PMA-qPCR)-for accurate and consistent differentiation between living and dead cells. In the practical experimental example, the number of genome copies representing living cells was determined in a mixed biofilm of <i>Candida albicans</i>-<i>Staphylococcus aureus</i> inhibited by photodynamic inactivation. Optimal conditions such as PMA concentration and the duration of light exposure, the optimization of DNA isolation from the mixed biofilm and standardization of PMA-qPCR parameters were tested prior to the main experiment. The genome copy number was calculated based on the known amount of genomic DNA in the qPCR and the genome size of the respective microorganism. The results showed that photodynamic inactivation in the presence of 1 mM methylene blue decreased the total genome copy number from 1.65 × 10⁸ to 3.19 × 10⁷, and from 4.39 × 10⁷ to 1.91 × 10⁷ for <i>S. aureus</i> and <i>C. albicans</i> (<i>P </i><<i> </i>0.01), respectively. The main disadvantage is the overestimation of the number of living cells represented by genome copy numbers. Such cells are unable to reproduce and grow (no vitality) and are continuously dying. On the other hand, PMA-qPCR determines the copy numbers of all microbial species, including a mix of eukaryotic yeasts and prokaryotic bacteria in a biofilm in one step, which is a great advantage.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae081"},"PeriodicalIF":2.5,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11631528/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142808244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}