Biology Methods and Protocols最新文献_第7页

Generating CRISPR-edited clonal lines of cultured Drosophila S2 cells. 从培养的果蝇 S2 细胞中产生 CRISPR 编辑克隆系。

IF 2.5

Biology Methods and Protocols Pub Date : 2024-08-17 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae059

John M Ryniawec, Anastasia Amoiroglou, Gregory C Rogers

{"title":"Generating CRISPR-edited clonal lines of cultured Drosophila S2 cells.","authors":"John M Ryniawec, Anastasia Amoiroglou, Gregory C Rogers","doi":"10.1093/biomethods/bpae059","DOIUrl":"https://doi.org/10.1093/biomethods/bpae059","url":null,"abstract":"CRISPR/Cas9 genome editing is a pervasive research tool due to its relative ease of use. However, some systems are not amenable to generating edited clones due to genomic complexity and/or difficulty in establishing clonal lines. For example, Drosophila Schneider 2 (S2) cells possess a segmental aneuploid genome and are challenging to single-cell select. Here, we describe a streamlined CRISPR/Cas9 methodology for knock-in and knock-out experiments in S2 cells, whereby an antibiotic resistance gene is inserted in-frame with the coding region of a gene-of-interest. By using selectable markers, we have improved the ease and efficiency for the positive selection of null cells using antibiotic selection in feeder layers followed by cell expansion to generate clonal lines. Using this method, we generated the first acentrosomal S2 cell lines by knocking-out centriole genes Polo-like Kinase 4/Plk4 or Ana2 as proof of concept. These strategies for generating gene-edited clonal lines will add to the collection of CRISPR tools available for cultured Drosophila cells by making CRISPR more practical and therefore improving gene function studies.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae059"},"PeriodicalIF":2.5,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11357795/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142113011","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}

引用次数: 0

Advancing age grading techniques for Glossina morsitans morsitans, vectors of African trypanosomiasis, through mid-infrared spectroscopy and machine learning. 通过中红外光谱仪和机器学习，推进非洲锥虫病传播媒介--莫西干蜱（Glossina morsitans morsitans）的年龄分级技术。

IF 2.5

Biology Methods and Protocols Pub Date : 2024-08-17 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae058

Mauro Pazmiño-Betancourth, Ivan Casas Gómez-Uribarri, Karina Mondragon-Shem, Simon A Babayan, Francesco Baldini, Lee Rafuse Haines

{"title":"Advancing age grading techniques for Glossina morsitans morsitans, vectors of African trypanosomiasis, through mid-infrared spectroscopy and machine learning.","authors":"Mauro Pazmiño-Betancourth, Ivan Casas Gómez-Uribarri, Karina Mondragon-Shem, Simon A Babayan, Francesco Baldini, Lee Rafuse Haines","doi":"10.1093/biomethods/bpae058","DOIUrl":"10.1093/biomethods/bpae058","url":null,"abstract":"Tsetse are the insects responsible for transmitting African trypanosomes, which cause sleeping sickness in humans and animal trypanosomiasis in wildlife and livestock. Knowing the age of these flies is important when assessing the effectiveness of vector control programs and modelling disease risk. Current methods to assess fly age are, however, labour-intensive, slow, and often inaccurate as skilled personnel are in short supply. Mid-infrared spectroscopy (MIRS), a fast and cost-effective tool to accurately estimate several biological traits of insects, offers a promising alternative. This is achieved by characterising the biochemical composition of the insect cuticle using infrared light coupled with machine-learning (ML) algorithms to estimate the traits of interest. We tested the performance of MIRS in estimating tsetse sex and age for the first-time using spectra obtained from their cuticle. We used 541 insectary-reared Glossina m. morsitans of two different age groups for males (5 and 7 weeks) and three age groups for females (3 days, 5 weeks, and 7 weeks). Spectra were collected from the head, thorax, and abdomen of each sample. ML models differentiated between male and female flies with a 96% accuracy and predicted the age group with 94% and 87% accuracy for males and females, respectively. The key infrared regions important for discriminating sex and age classification were characteristic of lipid and protein content. Our results support the use of MIRS as a rapid and accurate way to identify tsetse sex and age with minimal pre-processing. Further validation using wild-caught tsetse could pave the way for this technique to be implemented as a routine surveillance tool in vector control programmes.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae058"},"PeriodicalIF":2.5,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11407438/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142297453","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}

引用次数: 0

NanoMGT: Marker gene typing of low complexity mono-species metagenomic samples using noisy long reads. NanoMGT：使用噪声长读数对低复杂度单物种元基因组样本进行标记基因分型。

IF 2.5

Biology Methods and Protocols Pub Date : 2024-08-06 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae057

Malte B Hallgren, Philip T L C Clausen, Frank M Aarestrup

{"title":"NanoMGT: Marker gene typing of low complexity mono-species metagenomic samples using noisy long reads.","authors":"Malte B Hallgren, Philip T L C Clausen, Frank M Aarestrup","doi":"10.1093/biomethods/bpae057","DOIUrl":"https://doi.org/10.1093/biomethods/bpae057","url":null,"abstract":"Rapid advancements in sequencing technologies have led to significant progress in microbial genomics, yet challenges persist in accurately identifying microbial strain diversity in metagenomic samples, especially when working with noisy long-read data from platforms like Oxford Nanopore Technologies (ONT). In this article, we introduce NanoMGT, a tool designed to enhance marker gene typing in low-complexity mono-species samples, leveraging the unique properties of long reads. NanoMGT excels in its ability to accurately identify mutations amidst high error rates, ensuring the reliable detection of multiple strain-specific marker genes. Our tool implements a novel scoring system that rewards mutations co-occurring across different reads and penalizes densely grouped, likely erroneous variants, thereby achieving a good balance between sensitivity and precision. A comparative evaluation of NanoMGT, using a simulated multi-strain sample of seven bacterial species, demonstrated superior performance relative to existing tools and the advantages of using a threshold-based filtering approach to calling minority variants in ONT's sequencing data. NanoMGT's potential as a post-binning tool in metagenomic pipelines is particularly notable, enabling researchers to more accurately determine specific alleles and understand strain diversity in microbial communities. Our findings have significant implications for clinical diagnostics, environmental microbiology, and the broader field of genomics. The findings offer a reliable and efficient approach to marker gene typing in complex metagenomic samples.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae057"},"PeriodicalIF":2.5,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11387619/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142297456","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}

引用次数: 0

Automatic detection of fish scale circuli using deep learning. 利用深度学习自动检测鱼鳞环。

IF 2.5

Biology Methods and Protocols Pub Date : 2024-07-31 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae056

Nora N Hanson, James P Ounsley, Jason Henry, Kasim Terzić, Bruno Caneco

{"title":"Automatic detection of fish scale circuli using deep learning.","authors":"Nora N Hanson, James P Ounsley, Jason Henry, Kasim Terzić, Bruno Caneco","doi":"10.1093/biomethods/bpae056","DOIUrl":"10.1093/biomethods/bpae056","url":null,"abstract":"Teleost fish scales form distinct growth rings deposited in proportion to somatic growth in length, and are routinely used in fish ageing and growth analyses. Extraction of incremental growth data from scales is labour intensive. We present a fully automated method to retrieve this data from fish scale images using Convolutional Neural Networks (CNNs). Our pipeline of two CNNs automatically detects the centre of the scale and individual growth rings (circuli) along multiple radial transect emanating from the centre. The focus detector was trained on 725 scale images and achieved an average precision of 99%; the circuli detector was trained on 40 678 circuli annotations and achieved an average precision of 95.1%. Circuli detections were made with less confidence in the freshwater zone of the scale image where the growth bands are most narrowly spaced. However, the performance of the circuli detector was similar to that of another human labeller, highlighting the inherent ambiguity of the labelling process. The system predicts the location of scale growth rings rapidly and with high accuracy, enabling the calculation of spacings and thereby growth inferences from salmon scales. The success of our method suggests its potential for expansion to other species.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae056"},"PeriodicalIF":2.5,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11330318/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142000884","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}

引用次数: 0

Harmonizing immune cell sequences for computational analysis with large language models. 协调免疫细胞序列，利用大型语言模型进行计算分析。

IF 2.5

Biology Methods and Protocols Pub Date : 2024-07-30 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae055

Areej Alsaafin, Hamid R Tizhoosh

引用次数: 0

Simple protocol for combined extraction of exocrine secretions and RNA in small arthropods. 小型节肢动物外分泌分泌物和 RNA 联合提取的简单方案。

IF 2.5

Biology Methods and Protocols Pub Date : 2024-07-30 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae054

David Fröhlich, Michaela Bodner, Günther Raspotnig, Christoph Hahn

{"title":"Simple protocol for combined extraction of exocrine secretions and RNA in small arthropods.","authors":"David Fröhlich, Michaela Bodner, Günther Raspotnig, Christoph Hahn","doi":"10.1093/biomethods/bpae054","DOIUrl":"10.1093/biomethods/bpae054","url":null,"abstract":"The integration of data from multiple sources and analytical techniques to obtain novel insights and answer challenging questions is a hallmark of modern science. In arthropods, exocrine secretions may act as pheromones, defensive substances, antibiotics, as well as surface protectants, and as such they play a crucial role in ecology and evolution. Exocrine chemical compounds are frequently characterized by gas chromatography-mass spectrometry. Technological advances of recent years now allow us to routinely characterize the total gene complement transcribed in a particular biological tissue, often in the context of experimental treatment, via RNAseq. We here introduce a novel methodological approach to successfully characterize exocrine secretions and full transcriptomes of one and the same individual of oribatid mites. We found that chemical extraction prior to RNA extraction had only minor effects on the total RNA integrity. De novo transcriptomes obtained from such combined extractions were of comparable quality to those assembled for samples that were subject to RNA extraction only, indicating that combined chemical/RNA extraction is perfectly suitable for phylotranscriptomic studies. However, in-depth analysis of RNA expression analysis indicates that chemical extraction prior to RNAseq may affect transcript degradation rates, similar to the effects reported in previous studies comparing RNA extraction protocols. With this pilot study, we demonstrate that profiling chemical secretions and RNA expression levels from the same individual is methodologically feasible, paving the way for future research to understand the genes and pathways underlying the syntheses of biogenic chemical compounds. Our approach should be applicable broadly to most arachnids, insects, and other arthropods.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae054"},"PeriodicalIF":2.5,"publicationDate":"2024-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11316613/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141917564","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}

引用次数: 0

Deep learning pipeline reveals key moments in human embryonic development predictive of live birth after in vitro fertilization. 深度学习管道揭示了人类胚胎发育过程中可预测体外受精后活产的关键时刻。

IF 2.5

Biology Methods and Protocols Pub Date : 2024-07-19 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae052

Camilla Mapstone, Helen Hunter, Daniel Brison, Julia Handl, Berenika Plusa

引用次数: 0

The landscape of RNA 3D structure modeling with transformer networks. 利用变压器网络进行 RNA 3D 结构建模的前景。

IF 2.5

Biology Methods and Protocols Pub Date : 2024-07-02 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae047

Sumit Tarafder, Rahmatullah Roche, Debswapna Bhattacharya

引用次数: 0

Machine learning of cellular metabolic rewiring. 细胞代谢重新布线的机器学习

IF 2.5

Biology Methods and Protocols Pub Date : 2024-07-02 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae048

Joao B Xavier

{"title":"Machine learning of cellular metabolic rewiring.","authors":"Joao B Xavier","doi":"10.1093/biomethods/bpae048","DOIUrl":"10.1093/biomethods/bpae048","url":null,"abstract":"Metabolic rewiring allows cells to adapt their metabolism in response to evolving environmental conditions. Traditional metabolomics techniques, whether targeted or untargeted, often struggle to interpret these adaptive shifts. Here, we introduce MetaboLiteLearner, a lightweight machine learning framework that harnesses the detailed fragmentation patterns from electron ionization (EI) collected in scan mode during gas chromatography/mass spectrometry to predict changes in the metabolite composition of metabolically adapted cells. When tested on breast cancer cells with different preferences to metastasize to specific organs, MetaboLiteLearner predicted the impact of metabolic rewiring on metabolites withheld from the training dataset using only the EI spectra, without metabolite identification or pre-existing knowledge of metabolic networks. Despite its simplicity, the model learned captured shared and unique metabolomic shifts between brain- and lung-homing metastatic lineages, suggesting cellular adaptations associated with metastasis to specific organs. Integrating machine learning and metabolomics paves the way for new insights into complex cellular adaptations.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae048"},"PeriodicalIF":2.5,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11249387/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141621138","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}

引用次数: 0

High-throughput evaluation of hemolytic activity through precise measurement of colony and hemolytic zone sizes of engineered Bacillus subtilis on blood agar. 通过精确测量血琼脂上工程枯草芽孢杆菌的菌落和溶血区大小，高通量评估溶血活性。

IF 2.5

Biology Methods and Protocols Pub Date : 2024-06-27 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae044

Takahiro Bamba, Rina Aoki, Yoshimi Hori, Shu Ishikawa, Ken-Ichi Yoshida, Naoaki Taoka, Shingo Kobayashi, Hisashi Yasueda, Akihiko Kondo, Tomohisa Hasunuma

{"title":"High-throughput evaluation of hemolytic activity through precise measurement of colony and hemolytic zone sizes of engineered Bacillus subtilis on blood agar.","authors":"Takahiro Bamba, Rina Aoki, Yoshimi Hori, Shu Ishikawa, Ken-Ichi Yoshida, Naoaki Taoka, Shingo Kobayashi, Hisashi Yasueda, Akihiko Kondo, Tomohisa Hasunuma","doi":"10.1093/biomethods/bpae044","DOIUrl":"10.1093/biomethods/bpae044","url":null,"abstract":"Biosurfactants have remarkable characteristics, such as environmental friendliness, high safety, and excellent biodegradability. Surfactin is one of the best-known biosurfactants produced by Bacillus subtilis. Because the biosynthetic pathways of biosurfactants, such as surfactin, are complex, mutagenesis is a useful alternative to typical metabolic engineering approaches for developing high-yield strains. Therefore, there is a need for high-throughput and accurate screening methods for high-yield strains derived from mutant libraries. The blood agar lysis method, which takes advantage of the hemolytic activity of biosurfactants, is one way of determining their concentration. This method includes inoculating microbial cells onto blood-containing agar plates, and biosurfactant production is assessed based on the size of the hemolytic zone formed around each colony. Challenges with the blood agar lysis method include low experimental reproducibility and a lack of established protocols for high-throughput screening. Therefore, in this study, we investigated the effects of the inoculation procedure and media composition on the formation of hemolytic zones. We also developed a workflow to evaluate the number of colonies using robotics. The results revealed that by arranging colonies at appropriate intervals and measuring the areas of colonies and hemolytic rings using image analysis software, it was possible to accurately compare the hemolytic activity among several colonies. Although the use of the blood agar lysis method for screening is limited to surfactants exhibiting hemolytic activity, it is believed that by considering the insights gained from this study, it can contribute to the accurate screening of strains with high productivity.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae044"},"PeriodicalIF":2.5,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11219306/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141499181","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}

引用次数: 0