Current protocols最新文献

The endoplasmic reticulum (ER) is the main reservoir of Ca²⁺ of the cell. Accurate and quantitative measuring of Ca²⁺ dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca²⁺ indicators have been developed, including a family of fluorescent Ca²⁺ indicators, dubbed GFP-Aequorin Proteins (GAPs). They are based on the fusion of two jellyfish proteins, the green fluorescent protein (GFP) and the Ca²⁺-binding protein aequorin. GAP Ca²⁺ indicators exhibit a combination of several features: they are excitation ratiometric indicators, with reciprocal changes in the fluorescence excited at 405 and 470 nm, which is advantageous for imaging experiments; they exhibit a Hill coefficient of 1, which facilitates the calibration of the fluorescent signal into Ca²⁺ concentrations; they are insensible to variations in the Mg²⁺ concentrations or pH variations (in the 6.5-8.5 range); and, due to the lack of mammalian homologues, these proteins have a favorable expression in transgenic animals. A low Ca²⁺ affinity version of GAP, GAP3 (K_D ≅ 489 µM), has been engineered to conform with the estimated [Ca²⁺] in the ER. GAP3 targeted to the lumen of the ER (erGAP3) can be utilized for imaging intraluminal Ca²⁺. The ratiometric measurements provide a quantitative method to assess accurate [Ca²⁺]_ER, both dynamically and at rest. In addition, erGAP3 can be combined with synthetic cytosolic Ca²⁺ indicators to simultaneously monitor ER and cytosolic Ca²⁺. Here, we provide detailed methods to assess erGAP3 expression and to perform Ca²⁺ imaging, either restricted to the ER lumen, or simultaneously in the ER and the cytosol. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Detection of erGAP3 in the ER by immunofluorescence

Basic Protocol 2: Monitoring ER Ca²⁺

Basic Protocol 3: Monitoring ER- and cytosolic-Ca²⁺

Support Protocol: Generation of a stable cell line expressing erGAP3

Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb₃). Accumulation of Gb₃ in patients’ cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb₃ and creatinine. Samples are diluted with an internal standard solution containing Gb₃(C17:0) and creatinine-D₃, centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb₃ isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb₃ limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb₃. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb₃/creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Analysis of urinary globotriaosylceramide (Gb₃) and creatinine by UHPLC-MS/MS

Support Protocol 1: Preparation of the urinary quality controls

Support Protocol 2: Preparation of the urine matrix used for the Gb₃ calibration curve

Support Protocol 3: Preparation of the Gb₃ calibrators

Support Protocol 4: Preparation of the working solution containing the internal standards

Support Protocol 5: Preparation of the creatinine calibrators

Support Protocol 6: Preparation of the UHPLC solutions and mobile phases

Techno-functional properties of protein isolates such as emulsification, foaming, and gelling serve as key indicators to determine their food applications. Conventional macro-volume techniques used to measure these techno-functional properties are usually time consuming, require large amounts of protein samples, and are impractical when diverse protein samples are handled at the early screening stage. To overcome these issues, we have developed scaled-down (miniaturized) assays to test techno-functional properties of protein samples. These assays are simple, efficient, and require <400 μl of protein solution. Specifically, the miniaturized emulsification and gelling assays require 25-fold less protein than conventional macro-volume techniques and the miniaturized foaming assay requires 100-fold less sample. The performance of these assays has been thoroughly validated using conventional techno-functional tests for each parameter. The protocols described herein offer high-throughput screening capabilities, accelerating the testing process for protein techno-functional properties and allowing for quick identification of samples of interest from diverse samples. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Miniaturized emulsification assay

Alternate Protocol 1: Conventional macro-volume emulsification assay

Basic Protocol 2: Miniaturized foaming assay

Alternate Protocol 2: Conventional macro-volume foaming assay

Basic Protocol 3: Miniaturized gelling assay

Alternate Protocol 3: Conventional macro-volume gelling assay

U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites. Of significant interest, U1-70K has been implicated in Alzheimer's disease, in which it tends to form detergent-insoluble aggregates. Even though it was identified more than three decades ago, our understanding of U1-70K remains notably constrained, primarily due to challenges such as low levels of recombinant expression, susceptibility to protein degradation, and insolubility. In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Expression and purification of full-length U1-70K from E. coli

Support Protocol 1: Making chemically competent BL21 Star pRARE/pBB535 cells

Basic Protocol 2: Phosphorylation of full-length U1-70K using SRPK1

Support Protocol 2: Purification of SRPK1

Basic Protocol 3: Expression and purification of U1-70K BAD1 from E. coli

Basic Protocol 4: Phosphorylation of U1-70K BAD1 using SRPK1

Basic Protocol 5: Expression and purification of U1-70K BAD2 from E. coli

Molecular diagnostic point-of-care (MDx POC) testing is gaining momentum and is increasingly important for infectious disease detection and monitoring, as well as other diagnostic areas such as oncology. Molecular testing has traditionally required high-complexity laboratories. Laboratory testing complexity is determined by utilizing the Clinical Laboratory Improvement Amendments of 1988 (CLIA) Categorization Criteria scorecard, utilizing seven criteria that are scored on a scale of one to three. Previously, most commercially available point-of-care (POC) tests use other analytes and technologies that were not found to be highly complex by the CLIA scoring system. However, during the COVID-19 pandemic, MDx POC testing became much more prominent. Utilization during the COVID-19 pandemic has demonstrated that MDx POC testing applications can have outstanding advantages compared to available non-molecular POC diagnostic tests. This article introduces MDx POC testing to students, technologists, researchers, and others, providing a general algorithm for MDx POC test development. This algorithm is an introductory, step-by-step decision tree for defining a molecular POC diagnostic device meeting the functional requirements for a desired application. The technical considerations driving the decision-making include nucleic acid selection method (DNA, RNA), extraction methods, sample preparation, number of targets, amplification technology, and detection method. The scope of this article includes neither higher-order multiplexing, nor quantitative molecular analysis. This article covers key application considerations, such as sensitivity, specificity, turnaround time, and shipping/storage requirements. This article provides an overall understanding of the best resources and practices to use when developing a MDx POC assay that may be a helpful resource for readers without extensive molecular testing experience as well as for those who are already familiar with molecular testing who want to increase MDx availability at the POC. © 2024 Wiley Periodicals LLC.

Working memory capacity (WMC), a crucial component of working memory (WM), has consistently drawn the attention of researchers. Exploring the underlying neurobiological mechanisms behind it is currently a prominent focus in the field of neuroscience. Previously, we developed a novel behavioral paradigm for rodents called the olfactory working memory capacity (OWMC) paradigm, which serves as an effective tool for quantifying the WMC of rodents. The OWMC task comprises five phases: context adaptation, digging training, rule-learning for nonmatching to a single sample odor (NMSS), rule-learning for nonmatching to multiple sample odors (NMMS), and capacity testing. In the first phase, mice are handled to reduce stress and acclimate to the training cage. The second phase involves training mice to dig in a bowl of unscented sawdust to locate a piece of cheese. In the third phase, mice are trained to locate the cheese pellet in a bowl with a noveal odor. The fourth phase requires mice to distinguish the novel odor among multiple scented bowls to locate the cheese pellet. Finally, in the fifth phase, mice undergo several WMC tests until they achieve a stable level of performance. In this protocol paper, we will provide detailed instructions on how to implement the behavioral paradigm. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Context adaptation

Basic Protocol 2: Digging training

Basic Protocol 3: Rule-learning for NMSS

Basic Protocol 4: Rule-learning for NMMS

Basic Protocol 5: Capacity testing

Microbiome sequencing is at the forefront of health management development, and as such, it is becoming of great interest to monitor the microbiome in the aquaculture industry as well. Oxford Nanopore Technologies (ONT) platforms are gaining popularity to study microbial communities, enabling faster sequencing, extended read length, and therefore, improved taxonomic resolution. Despite this, there is a lack of clear guidelines to perform a metabarcoding study, especially when dealing with samples from non-mammalian species, such as aquaculture-related samples. In this article, we provide general guidelines for sampling, nucleic acid extraction, and ONT-based library preparation for both environmental (water, sediment) and host-associated (gill or skin mucus, skin, gut content, or gut mucosa) microbiome analysis. Our procedures focus specifically on rainbow trout (Oncorhynchus mykiss) reared in experimental facilities. However, these protocols can also be transferred to alternative types of samples, such as environmental DNA (eDNA) monitoring from alternative water sources, or to different fish species. The additional challenge posed by the low biomass and limited bacterial diversity inherent in fish-associated microbiomes is addressed through the implementation of troubleshooting solutions. Furthermore, we describe a bioinformatic pipeline starting from raw reads and leading to taxonomic abundance tables using currently available tools and software. Finally, we provide a set of specific guidelines and considerations related to the strategic planning of a microbiome study within the context of aquaculture. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Environmental sample collection

Basic Protocol 2: Host-associated sample collection

Alternate Protocol: Host-associated sample collection: Alternative sample types

Basic Protocol 3: Sample pre-treatment and nucleic acid extraction

Basic Protocol 4: Quality control and preparation for 16S rRNA gene sequencing

Support Protocol 1: Assessment of inhibition by quantitative PCR

Support Protocol 2: Bioinformatic analysis from raw files to taxonomic abundance tables

The microtubule (MT) cytoskeleton performs a variety of functions in cell division, cell architecture, neuronal differentiation, and ciliary beating. These functions are controlled by proteins that directly interact with MTs, commonly referred to as microtubule-associated proteins (MAPs). Out of the many proteins reported interact with MTs, only a some have been biochemically and functionally characterized so far. One of the limitations of classical in vitro assays and single-MT reconstitution approaches is that they are typically performed with purified proteins. As purification of proteins can be difficult and time-consuming, many previous studies have only focused on a few proteins, while systematic analyses of many different proteins by in vitro reconstitution assays were not possible. Here we present a detailed protocol using lysates of mammalian cells instead of purified proteins that overcomes this limitation. Those lysates contain all molecular components required for in vitro MT reconstitution including the endogenous tubulin and the recombinant MAPs, which form MT assemblies upon the injection of the lysates into a microscopy chamber. This allows to directly observe the dynamic behavior of growing MTs, as well as the fluorescently labeled associated proteins by total internal reflection fluorescence (TIRF) microscopy. Strikingly, all proteins tested so far were functional in our approach, thus providing the possibility to test virtually any protein of interest. This also opens the possibility to screen the impact of patient mutations on the MT binding behavior of MAPs in a medium-throughput manner. In addition, the lysate approach can easily be adapted to other applications that have predominantly been performed with purified proteins so far, such as investigating other cytoskeletal systems and cytoskeletal crosstalk, or to study structures of MAPs bound to MTs by cryo-electron microscopy. Our approach is thus a versatile, expandable, and easy-to-use method to characterize the impact of a broad spectrum of proteins on cytoskeletal behavior and function. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Preparation of lysates of human cells for TIRF reconstitution assays

Basic Protocol 2: Quantification of GFP-tagged MAP concentration in cell lysates

Support Protocol 1: Purification of KIF5B(N555/T92A) (dead kinesin) protein for TIRF reconstitution assays

Support Protocol 2: Preparation of GMPCPP MT seeds for TIRF reconstitution assays

Basic Protocol 3: TIRF-based MT-MAP reconstitution assays using cell lysates

Sequence changes in viral genomes generate protein sequence diversity that enables viruses to evade the host immune system, hindering the development of effective preventive and therapeutic interventions. The massive proliferation of sequence data provides unprecedented opportunities to study viral adaptation and evolution. An alignment-free approach removes various restrictions posed by an alignment-dependent approach for studying sequence diversity. The publicly available tool, UNIQmin, offers an alignment-free approach for studying viral sequence diversity at any given rank of taxonomy lineage and is big data ready. The tool performs an exhaustive search to determine the minimal set of sequences required to capture the peptidome diversity within a given dataset. This compression is possible through the removal of identical sequences and unique sequences that do not contribute effectively to the peptidome diversity pool. Herein, we describe a detailed four-part protocol utilizing UNIQmin to generate the minimal set for the purpose of viral diversity analyses, alignment-free at any rank of the taxonomy lineage, using the recent global public health threat Monkeypox virus (MPX) sequence data as a case study. The protocol enables a systematic bioinformatics approach to study sequence diversity across taxonomic lineages, which is crucial for our future preparedness against viral epidemics. This is particularly important when data are abundant, freely available, and alignment is not an option. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Tool installation and input file preparation

Basic Protocol 2: Generation of a minimal set of sequences for a given dataset

Basic Protocol 3: Comparative minimal set analysis across taxonomic lineage ranks

Basic Protocol 4: Factors affecting the minimal set of sequences

The European Bioinformatics Institute (EMBL-EBI)’s Job Dispatcher framework provides access to a wide range of core databases and analysis tools that are of key importance in bioinformatics. As well as providing web interfaces to these resources, web services are available using REST and SOAP protocols that enable programmatic access and allow their integration into other applications and analytical workflows and pipelines. This article describes the various options available to researchers and bioinformaticians who would like to use our resources via the web interface employing RESTful web services clients provided in Perl, Python, and Java or who would like to use Docker containers to integrate the resources into analysis pipelines and workflows. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Retrieving data from EMBL-EBI using Dbfetch via the web interface

Alternate Protocol 1: Retrieving data from EMBL-EBI using WSDbfetch via the REST interface

Alternate Protocol 2: Retrieving data from EMBL-EBI using Dbfetch via RESTful web services with Python client

Support Protocol 1: Installing Python REST web services clients

Basic Protocol 2: Sequence similarity search using FASTA search via the web interface

Alternate Protocol 3: Sequence similarity search using FASTA via RESTful web services with Perl client

Support Protocol 2: Installing Perl REST web services clients

Basic Protocol 3: Sequence similarity search using NCBI BLAST+ RESTful web services with Python client

Basic Protocol 4: Sequence similarity search using HMMER3 phmmer REST web services with Perl client and Docker

Support Protocol 3: Installing Docker and running the EMBL-EBI client container

Basic Protocol 5: Protein functional analysis using InterProScan 5 RESTful web services with the Python client and Docker

Alternate Protocol 4: Protein functional analysis using InterProScan 5 RESTful web services with the Java client

Support Protocol 4: Installing Java web services clients

Basic Protocol 6: Multiple sequence alignment using Clustal Omega via web interface

Alternate Protocol 5: Multiple sequence alignment using Clustal Omega with Perl client and Docker

Support Protocol 5: Exploring the RESTful API with OpenAPI User Inferface