Journal of biological methods最新文献_第5页

Utilizing a human TLR selective ligand in a humanized immune system mouse model to investigate human TLR4 signaling. 利用人TLR选择性配体在人源化免疫系统小鼠模型中研究人TLR4信号转导。

Journal of biological methods Pub Date : 2023-11-20 eCollection Date: 2023-01-01 DOI: 10.14440/jbm.2023.408

Rachel Twomey, Sean Graham, Joseph S Spina, Xiaoming Wu, Philip E Dubé, Courtney Ferrebee, William Housley

{"title":"Utilizing a human TLR selective ligand in a humanized immune system mouse model to investigate human TLR4 signaling.","authors":"Rachel Twomey, Sean Graham, Joseph S Spina, Xiaoming Wu, Philip E Dubé, Courtney Ferrebee, William Housley","doi":"10.14440/jbm.2023.408","DOIUrl":"10.14440/jbm.2023.408","url":null,"abstract":"Mouse models with humanized immune systems are becoming increasingly prevalent in pharmaceutical research as a platform for preclinical testing with potential for greater translatability to clinical applications. However, the presence of both mouse and human cells that respond to TLR ligands poses a challenge for investigating therapeutic modalities targeting TLR signaling. AZ617 is a human TLR4 agonist, which has been shown in vitro to preferentially induce human cytokines via the TLR4 signaling pathway. We sought to examine the ability of AZ617 to preferentially induce human cytokines in CD34+ stem cell-engrafted NOG-EXL mice (huNOG-EXL), to determine its suitability as an in vivo human functional readout. AZ617 elicited a strong human TNFα and IL-6 response in vivo that demonstrated a 10- and 5-fold preference, respectively, over the mouse TNFα and IL-6. To assess efficacy of inhibiting a key protein in the TLR4 signaling pathway, PF-06650833, a small molecule inhibitor of IRAK4, was used as a tool molecule. PF-0660833 was found to effectively inhibit AZ617-induced human TNFα release in vitro. Likewise, PF-06650833 reduced AZ617-induced human TNFα in the huNOG-EXL mouse model, with a weaker effect on human IL-6. A longitudinal study tracking functionality of monocytes revealed that the ability of monocytes to respond to ex vivo stimuli was increased by 21 weeks after engraftment. Taken together, our data suggests that human selective TLR ligands could preferentially drive cytokine production from human cells in huNOG-EXL mice. This model will allow for investigation of pharmacological inhibition of human TLR signaling pathways in an in vivo model system.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"10 ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10691501/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138479727","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

A strategy to recover a poor-quality ligase product 一种回收劣质连接酶产品的策略

Journal of biological methods Pub Date : 2023-11-10 DOI: 10.14440/jbm.2023.411

Sonia Del Prete, Marta Gogliettino, Gianna Palmieri, Ennio Cocca

{"title":"A strategy to recover a poor-quality ligase product","authors":"Sonia Del Prete, Marta Gogliettino, Gianna Palmieri, Ennio Cocca","doi":"10.14440/jbm.2023.411","DOIUrl":"https://doi.org/10.14440/jbm.2023.411","url":null,"abstract":"Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation.We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5’ end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"98 8","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135091708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Optimizing factors for large-scale production of Arbuscular Mycorrhizal Fungi consortia using root organ cultures 利用根器官培养大规模生产丛枝菌根真菌的优化因素

Journal of biological methods Pub Date : 2023-11-08 DOI: 10.14440/jbm.2023.410

Maunata Ghorui, Shouvik Chowdhury, Keshab Das, KIRAN SUNAR, Balu Prakash

{"title":"Optimizing factors for large-scale production of Arbuscular Mycorrhizal Fungi consortia using root organ cultures","authors":"Maunata Ghorui, Shouvik Chowdhury, Keshab Das, KIRAN SUNAR, Balu Prakash","doi":"10.14440/jbm.2023.410","DOIUrl":"https://doi.org/10.14440/jbm.2023.410","url":null,"abstract":"Large-scale production of Arbuscular Mycorrhizal Fungi (AMF) consortia is a crucial stride in harnessing their potential for sustainable agriculture and plant growth enhancement. However, establishing optimal production conditions is challenging due to their obligate nature, variability, lack of standardized protocols, and limited understanding of their specific requirements. Previous attempts to standardize Root Organ Cultures (ROC) for AMF overlooked challenges related to viable inoculum production for field applications. This current investigation reported, for the first time, the optimization of various factors during large-scale production of AMF using ROC. By optimizing factors like gelling agents, media preparation, medium-to-inoculum ratios, incubation conditions, age, harvesting method and drying temperatures, we achieved significant yields of viable propagules. The standardized protocol outlined in this study will greatly influence commercial-scale AMF production. These standardized protocols are poised to contribute to larger-scale AMF production worldwide, with the potential to support sustainable agriculture and ecosystem management.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" 39","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135340909","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Prevention, Diagnosis and Eradication of Mycoplasma Contamination in Cell Culture 细胞培养中支原体污染的预防、诊断和根除

Journal of biological methods Pub Date : 2023-11-01 DOI: 10.14440/jbm.2023.407

Xuefeng Huang, Minghang Yu, Bingbing Wang, Yanlong Zhang, Junjing Xue, Yu Fu, Xi Wang

引用次数: 0

An experimental workflow for identifying RNA m6A alterations in cellular senescence by methylated RNA immunoprecipitation sequencing 通过甲基化RNA免疫沉淀测序鉴定细胞衰老中RNA m6A变化的实验流程

Journal of biological methods Pub Date : 2023-08-25 DOI: 10.14440/jbm.2023.403

Yue Shi, Zeming Wu, Weiqi Zhang, J. Qu, W. Ci, Guang-Hui Liu

引用次数: 0

Benchmarking assembly free nanopore read mappers to classify complex millipede gut microbiota via Oxford Nanopore Sequencing Technology. 通过牛津纳米孔测序技术对无组装纳米孔读数映射器进行基准测试，以对复杂的千足虫肠道微生物群进行分类

Journal of biological methods Pub Date : 2023-08-04 eCollection Date: 2023-01-01 DOI: 10.14440/jbm.2023.376

Orlando J Geli-Cruz, Carlos J Santos-Flores, Matias J Cafaro, Alex Ropelewski, Alex R Van Dam

{"title":"Benchmarking assembly free nanopore read mappers to classify complex millipede gut microbiota via Oxford Nanopore Sequencing Technology.","authors":"Orlando J Geli-Cruz, Carlos J Santos-Flores, Matias J Cafaro, Alex Ropelewski, Alex R Van Dam","doi":"10.14440/jbm.2023.376","DOIUrl":"10.14440/jbm.2023.376","url":null,"abstract":"Millipedes are key players in recycling leaf litter into soil in tropical ecosystems. To elucidate their gut microbiota, we collected millipedes from different municipalities of Puerto Rico. Here we aim to benchmark which method is best for metagenomic skimming of this highly complex millipede microbiome. We sequenced the gut DNA with Oxford Nanopore Technologies' (ONT) MinION sequencer, then analyzed the data using MEGAN-LR, Kraken2 protein mode, Kraken2 nucleotide mode, GraphMap, and Minimap2 to classify these long ONT reads. From our two samples, we obtained a total of 87,110 and 99,749 ONT reads, respectively. Kraken2 nucleotide mode classified the most reads compared to all other methods at the phylum and class taxonomic level, classifying 75% of the reads in the two samples, the other methods failed to assign enough reads to either phylum or class to yield asymptotes in the taxa rarefaction curves indicating that they required more sequencing depth to fully classify this community. The community is hyper diverse with all methods classifying 20-50 phyla in the two samples. There was significant overlap in the reads used and phyla classified between the five methods benchmarked. Our results suggest that Kraken2 nucleotide mode is the most appropriate tool for the application of metagenomic skimming of this highly complex community.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":"e99010003"},"PeriodicalIF":0.0,"publicationDate":"2023-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10627078/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48590579","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

Proximity Protein Labeling In Dictyostelium With Engineered Ascorbic Acid Peroxidase 2. 用工程抗坏血酸过氧化物酶 2 在竹荪中进行近距离蛋白质标记。

Journal of biological methods Pub Date : 2023-03-16 eCollection Date: 2023-01-01 DOI: 10.14440/jbm.2023.396

Jamie A Takashima, Helena A Woroniecka, Pascale G Charest

引用次数: 0

An experimental workflow for enrichment of low abundant proteins from human serum for the discovery of serum biomarkers. 从人血清中富集低丰度蛋白的实验流程，用于发现血清生物标志物。

Journal of biological methods Pub Date : 2023-01-01 DOI: 10.14440/jbm.2023.394

Mehmet Sarihan, Merve Gulsen Bal Albayrak, Murat Kasap, Gurler Akpinar, Elifcan Kocyigit

{"title":"An experimental workflow for enrichment of low abundant proteins from human serum for the discovery of serum biomarkers.","authors":"Mehmet Sarihan, Merve Gulsen Bal Albayrak, Murat Kasap, Gurler Akpinar, Elifcan Kocyigit","doi":"10.14440/jbm.2023.394","DOIUrl":"https://doi.org/10.14440/jbm.2023.394","url":null,"abstract":"Serum contains proteins that possess important information about diseases and their progression. Unfortunately, these proteins, which carry the information in the serum are in low abundance and are masked by other serum proteins that are in high abundance. Such masking prevents their identification and quantification. Therefore, removal of high abundance proteins is required to enrich, identify, and quantify the low abundance proteins. Immunodepletion methods are often used for this purpose, but there are limitations in their use because of off-target effects and high costs. Here we presented a robust, reproducible and cost-effective experimental workflow to remove immunoglobulins and albumin from serum with high efficiency. The workflow did not suffer from such limitations and enabled identification of 681 low abundance proteins that were otherwise undetectable in the serum. The identified low abundance proteins belonged to 21 different protein classes, namely the immunity-related proteins, modulators of protein-binding activity, and protein-modifying enzymes. They also played roles in various metabolic events, such as integrin signalling, inflammation-mediated signalling, and cadherin signalling. The presented workflow can be adapted to remove abundant proteins from other types of biological material and to provide considerable enrichment for low-abundance proteins.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"10 ","pages":"e99010001"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/db/0b/jbm-10-e99010001.PMC10062475.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9610553","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: 2

Estimating somatic mutation rates by bottlenecked duplex sequencing in non-model organisms: Daphnia magna as a case study. 通过瓶颈双链测序估算非模式生物的体细胞突变率：以大型水蚤为例。

Journal of biological methods Pub Date : 2022-11-03 eCollection Date: 2022-01-01 DOI: 10.14440/jbm.2022.391

Eli Sobel, Jeremy E Coate, Sarah Schaack

{"title":"Estimating somatic mutation rates by bottlenecked duplex sequencing in non-model organisms: Daphnia magna as a case study.","authors":"Eli Sobel, Jeremy E Coate, Sarah Schaack","doi":"10.14440/jbm.2022.391","DOIUrl":"10.14440/jbm.2022.391","url":null,"abstract":"Somatic mutations are evolutionarily important as determinants of individual organismal fitness, as well as being a focus of clinical research on age-related disease, such as cancer. Identifying somatic mutations and quantifying mutation rates, however, is extremely challenging and genome-wide somatic mutation rates have only been reported for a few model organisms. Here, we describe the application of Duplex Sequencing on bottlenecked WGS libraries to quantify somatic nuclear genome-wide base substitution rates in Daphnia magna. Daphnia, historically an ecological model system, has more recently been the focus of mutation studies, in part because of its high germline mutation rates. Using our protocol and pipeline, we estimate a somatic mutation rate of 5.6 × 10-7 substitutions per site (in a genotype where the germline rate is 3.60 × 10-9 substitutions per site per generation). To obtain this estimate, we tested multiple dilution levels to maximize sequencing efficiency and developed bioinformatic filters needed to minimize false positives when a high-quality reference genome is not available. In addition to laying the groundwork for estimating genotypic variation in rates of somatic mutations within D. magna, we provide a framework for quantifying somatic mutations in other non-model systems, and also highlight recent innovations to single molecule sequencing that will help to further refine such estimates.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"9 3","pages":"e165"},"PeriodicalIF":0.0,"publicationDate":"2022-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/68/ec/jbm-9-3-e165.PMC10040303.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9213300","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

Monobiotinylated Proteins Tethered to Microspheres for Detection of Antigen-Specific Serum Antibodies. 单生物素化蛋白拴在微球上检测抗原特异性血清抗体。

Journal of biological methods Pub Date : 2022-09-23 eCollection Date: 2021-01-01 DOI: 10.14440/jbm.2022.390

Caleb S Whitley, Thomas C Mitchell

{"title":"Monobiotinylated Proteins Tethered to Microspheres for Detection of Antigen-Specific Serum Antibodies.","authors":"Caleb S Whitley, Thomas C Mitchell","doi":"10.14440/jbm.2022.390","DOIUrl":"10.14440/jbm.2022.390","url":null,"abstract":"Surface modified microspheres have been leveraged as a useful way to immobilize antigen for serological studies. The use of carboxyl modified microspheres for this purpose is well-established, but commonly associated with technical challenges. Streptavidin modified microspheres require little technical expertise and thus address some of the shortcomings of carboxyl microspheres. An additional feature of streptavidin microspheres is the use of mono-biotinylated proteins, which contain a single biotinylation motif at the C-terminus. However, the relative performance of streptavidin and carboxyl microspheres is unknown. Here, we performed a head-to-head comparison of streptavidin and carboxyl microspheres. We compared antigen binding, orientation, and staining quality and found that both microspheres perform similarly based on these defined parameters. We also evaluated the utility of streptavidin microspheres bound to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD), to reliably detect RBD-specific IgG1, IgG3, and IgA1 produced in individuals recently immunized with Pfizer/BioNTech mRNA coronavirus disease (COVID) vaccine as 'proof-of-concept'. We provide evidence that each of the antibody targets are detectable in serum using RBD-coated microspheres, Ig-specific 'detector' monoclonal antibodies (mAbs), and flow cytometry. We found that cross-reactivity of the detector mAbs can be minimized by antibody titration to improve differentiation between IgG1 and IgG3. We also coated streptavidin microspheres with SARS-CoV-2 delta variant RBD to determine if the streptavidin microsphere approach revealed any differences in binding of immune serum antibodies to wild-type (Wuhan) versus variant RBD (Delta). Overall, our results show that streptavidin microspheres loaded with mono-biotinylated antigen is a robust alternative to chemically cross-linking antigen to carboxyl microspheres for use in serological assays.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 ","pages":"e164"},"PeriodicalIF":0.0,"publicationDate":"2022-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d7/01/jbm-8-specissue-e164.PMC9682163.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40708355","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