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At least 667 records · Page 37Linked to original sources

PubMed2004

N-t-butyliodoacetamide and iodoacetanilide: two new cysteine alkylating reagents for relative quantitation of proteins.

The synthesis and application of two new alkylating reagents, N-tert-butyl-2-iodoacetamide (N-t-butyliodoacetamide) and 2-iodo-N-phenylacetamide (iodoacetanilide), are described. N-t-Butyliodoacetamide and iodoacetanilide were synthesised to purity in their d(0)-light and in their respective d(9)- and d(5)-heavy forms. The newly synthesised reagents are covalently bound to peptides containing cysteines via an alkylation reaction. The mass differences of 5 and 9 Da avoid possible problems of overlapping isotope distribution. For each alkylated cysteine a peptide mass increases, respectively, by a multiple of 113 and 133 Da for the d(0)-light form of N-t-butyliodoacetamide and iodoacetanilide. These reagents can therefore replace common alkylating reagents in existing proteomics-based applications. Alkylated peptides increase in mass in the same mass range as amino acids and remain suitable for tandem mass spectrometry (MS/MS) data acquisition and analysis. The compounds are simple to use and derivatisation is based on widely applied alkylating procedures. Preliminary results show that these reagents can be applied for both protein quantitation and identification by peptide mass finger printing and/or MS/MS techniques. Using these chemicals and the suggested workflow enables the quantitative analysis of the whole protein sample and realises access to peptides that may contain potential post-translational modifications. Other approaches that incorporate a matrix-assisted laser desorption/ionisation (MALDI) interface prior to MS can take advantage of these chemicals, such as the molecular scanner.

Acetanilides↗

PubMed2005

Average peptide score: a useful parameter for identification of proteins derived from database searches of liquid chromatography/tandem mass spectrometry data.

The quantity and variable quality of data that can be generated from liquid chromatography (LC)/mass spectrometry (MS)-based proteomics analyses creates many challenges in interpreting the spectra in terms of the actual proteins in a complex sample. In spite of improvements in algorithms that match putative peptide sequences to MS/MS spectra, the assembly of these lists of possible or probable peptides into a 'correct' set of proteins is still problematic. We have observed a trend in a simple relationship, derived from standard database search outputs, which can be useful in assessing the quality of a MS/MS-based protein identification. Specifically, the ratio of the protein score and number of non-redundant peptides, or average peptide score (APS), can facilitate initial filtering of database search results in addition to providing a useful measure of confidence for the proteins identified. This parameter has been applied to results from the analysis of multi-protein complexes derived from pull-down experiments analyzed using a two-dimensional LC/MS/MS workflow. In particular, the complex list of protein identifications derived from a drug affinity pull-down with immobilized ampicillin and an E. coli lysate was greatly simplified by applying the APS as a filter, allowing for facile identification of the penicillin-binding proteins known to interact with ampicillin. Furthermore, an APS threshold can be used for any data sets derived from electrospray ionization (ESI)- or matrix-assisted laser desorption/ionization (MALDI)-MS/MS experiments and is also not specific to any database search program.

Chromatography, Liquid↗

PubMed2025

Nascent and Mature RNA Profiling by Subcellular Fractionation in Human Cells.

Transcription and RNA decay determine steady-state RNA levels in cells available for translation and RNA-mediated regulatory functions. Both processes can be assessed by various techniques, for majority, based on RNA labelling or chromatin immunoprecipitation, but require a high level of expertise. Here, we describe a cost-effective, fast, and simple protocol that enables the profiling of nascent and mature RNA in the cytoplasm, nucleoplasm, and chromatin through subcellular fractionation. The workflow can include α-amanitin inhibition of RNA Polymerase II to assess nascent RNAs as a proxy of transcriptional activity, or it can be used without this treatment to investigate distribution of partially processed or mature transcripts across distinct subcellular compartments. It is applicable for studying any of RNA biotypes, including small and long noncoding RNAs, mRNAs, and their splice variants, on both transcript-specific and transcriptome-wide scales. Nascent or mature RNAs isolated from each fraction can be further analyzed by any technique of choice (northern blot, reverse transcription, RNA sequencing).

Humans↗

PubMed2025

SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.

Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.

Humans↗

PubMed2025

Quantitative Profiling of Histone Variants and Posttranslational Modifications by Tandem Mass Spectrometry in Arabidopsis.

Histone dynamics constitute an important layer of gene regulations associated with development and growth in multicellular eukaryotes. They also stand as key determinants of plant responses to environmental changes. Histone dynamics include the exchange of histone variants as well as post-translational modifications of their amino acid residues (such as acetylation and mono/di/trimethylation), commonly referred to as histone marks. Investigating histone dynamics with a focus on combinatorial changes occurring at their residues will greatly help unravel how plants achieve phenotypic plasticity.Mass spectrometry (MS) analysis offers unequaled resolution of the abundance of histone variants and of their marks. Indeed, relative to other techniques such as western blot or genome-wide profiling, this powerful technique allows quantifying the relative abundances of histone forms, as well as revealing coexisting marks on the same histone molecule. Yet, while MS-based histone analysis has proven efficient in several animals and other model organisms, this method stands out as more challenging in plants. One major challenge is the isolation of sufficient amounts of pure, high-quality histones, likely rendered difficult by the presence of the cell wall, for sufficiently deep and resolutive identification of histone species.In this chapter, we describe a straightforward MS-based proteomic method, implemented to characterize histone marks from Arabidopsis thaliana seedling tissues and cell culture suspensions. After acid extraction of histones, in vitro propionylation of free lysine residues, and digestion with trypsin, a treatment at highly basic pH allows obtaining sharp spectral signals of biologically relevant histone peptide forms.The method workflow described here shall be used to measure changes in histone marks between Arabidopsis thaliana genotypes, along developmental time-courses, or upon various stresses and treatments.

Histones↗

PubMed2025

ChIP-Rx: Arabidopsis Chromatin Profiling Using Quantitative ChIP-Seq.

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is widely used to probe the chromatin landscape of transcription factors, chromatin components, and associated proteins. Conventional ChIP normalization procedures robustly allow estimating differences in local enrichment across genomic regions. Yet, inter-sample comparisons can be biased by technical variability and biological differences. This is notably the case when samples display large differences in the abundance of the target protein or its enrichment at chromatin. For example, epigenome defects are improperly detected or quantified upon large-effect genetic or chemical inhibition of chromatin modifiers. To circumvent these caveats and robustly determine biological variations while minimizing technical variability, ChIP adaptations using an external reference have flourished. Here, we describe a step-by-step protocol employing a reference exogenous chromatin (ChIP-Rx) that allows absolute comparisons of epigenome variations in Arabidopsis samples displaying drastic differences in chromatin mark abundance. In contrast to the originally published ChIP-Rx approach, which assumes that exogenous spike-in references are constant across samples, the method detailed here involves the sequencing of each input sample to account for technical variability in initial reference chromatin contents. We also report a detailed computational workflow with an accompanying Github resource to help in calculating spike-in normalization factors, applying them to normalize epigenome tracks, and performing spike-in normalized inter-sample differential analyses. We propose two ways of computing the spike-in factor: a classically used method based on raw counts and a noise-corrected method using peak detection on the exogenous genome.

Arabidopsis↗

PubMed2027

Global Genomic Surveillance.

Global genomic surveillance has emerged as a foundational pillar of public health in the twenty-first century, enabling real-time tracking of pathogen evolution and informing outbreak response. This chapter examines the strategic architecture of global genomic surveillance, focusing on its application to arboviruses such as chikungunya virus (CHIKV). It explores the integration of genomic data with epidemiological, clinical, and environmental information within a One Health framework, while addressing critical challenges in governance, equity, and interoperability. The discussion covers the entire genomic surveillance workflow, from sample collection and sequencing to bioinformatic analysis and phylogenetic inference, and highlights the transformative role of artificial intelligence (AI) in predictive surveillance. By analyzing global initiatives, operational barriers, and emerging technologies, this chapter underscores the necessity of sustainable, equitable, and interoperable genomic systems to proactively address current and future infectious disease threats.

Humans↗

PubMed2027

Optimized Amplicon Strategy for Long-Read Sequencing of the Chikungunya Virus Genome.

Chikungunya virus (CHIKV) is a positive-sense RNA alphavirus transmitted to humans primarily by Aedes aegypti and Aedes albopictus mosquitoes. Its global circulation and significant public health impact underscore the need to better understand the molecular mechanisms driving CHIKV pathogenesis and transmission. Although robust molecular biology methods exist for CHIKV genome sequencing, a major limitation for surveillance and research is the inability to determine whether two nucleotide variations co-occur within the same viral genome when they are separated beyond the span of typical short-read designs. Here, we describe an optimized approach for processing CHIKV RNA samples that generates large amplicons suitable for long-read nanopore sequencing. This protocol enables amplification of the complete CHIKV genome in only two or three amplicons and facilitates detection of co-occurring nucleotide variations across 4-7.5 kb within the same molecule, thereby simplifying sequencing workflows and improving resolution in studies of viral evolution.

Chikungunya virus↗

PubMed2027

Protocol for Detecting and Sequencing Chikungunya Virus from Field-Collected Mosquitoes.

Arboviral diseases represent a major public health challenge, especially in tropical regions where environmental conditions may favor the proliferation and spread of mosquito vectors. Thus, early and accurate detection of chikungunya virus (CHIKV) in mosquito populations can be a valuable tool for effective surveillance of circulating variants and for identifying new viral introductions. Given the challenges of detecting arboviruses in field-captured mosquitoes, we describe an integrated workflow for CHIKV molecular detection and whole-genome sequencing. This protocol includes mosquito homogenization using a bead-based mechanical disruptor, RNA extraction using TRIzol reagent with minor modifications, molecular screening using CHIKV-specific RT-qPCR, and whole-genome amplification followed by sequencing on Illumina platforms. Despite the protocol being optimized for individual mosquitoes, it results in high-quality RNA suitable for both entomological surveillance and genomic analysis. As this protocol allows recovery of complete CHIKV genomes from mosquito specimens, it can serve as a basis for genomic epidemiology studies, enabling monitoring of viral diversity and lineage dynamics, and facilitating early detection of emerging variants to support timely and targeted public health interventions in endemic and at-risk regions.

Animals↗

PubMed2027

Inferring Gene Regulatory Networks in Stem Cells: Methods and Applications.

Gene regulatory networks (GRNs) represent the complex interplay of transcription factors, regulatory elements, and target genes that orchestrate cellular identity and function, playing a crucial role in the differentiation and maintenance of stem cells. This chapter provides an overview of experimental and computational methodologies for inferring GRNs, with particular emphasis on single-cell approaches. We first review key experimental techniques for detecting transcription factor binding sites, chromatin accessibility, and DNA motifs, alongside essential databases that support GRN reconstruction. We then introduce computational inference methods that can be categorized into four principal frameworks: correlation-based approaches, regression and machine learning models, probabilistic and deep learning methods, and integrative or message-passing frameworks. To illustrate practical application, we present a case study applying the pySCENIC workflow to a peripheral blood mononuclear cell single-cell RNA sequencing dataset from mouse, demonstrating how regulon-based analysis can reveal cell-type-specific regulatory programs. This chapter aims to serve as a practical guide for researchers seeking to understand and implement GRN inference methodologies in stem cell biology and related fields.

Gene Regulatory Networks↗

PubMed2027

Systematic Analysis of Tumor Microenvironment Using IOBR.

The Immuno-Oncology Biological Research (IOBR) package is an R-based analysis tool for exploring the tumor microenvironment (TME) and its influence on anti-tumor immunity. Built for high-throughput data-spanning both transcriptomic and genomic profiles-IOBR integrates six analytical modules, including transcriptomic data preprocessing, TME profiling, TME pattern identification, ligand-receptor interaction analysis, genome-TME interaction assessment, and visualization. In this chapter, we walk through a multi-omics workflow using example datasets, illustrating data preparation, distribution analyses, result interpretation, and graphical output. IOBR is open source and is available at https://github.com/IOBR/IOBR and a detailed GitBook ( https://iobr.github.io/book/ ) offers a complete manual and analysis guide for each function.

Tumor Microenvironment↗

PubMed2027

Identification of Genome-Wide Chromatin Structural Aberration in Cancer by Hi-C Analysis.

Aberrant three-dimensional genome organization is a hallmark of cancer, often driving oncogene activation through mechanisms such as enhancer hijacking. High-throughput chromosome conformation capture (Hi-C) maps these interactions on a genome-wide scale. Unlike earlier dilution-based methods, in situ Hi-C performs proximity ligation within intact nuclei, minimizing random ligation noise and enabling fine-scale structure detection. This chapter describes an optimized in situ Hi-C protocol tailored for cancer cell lines using MboI digestion and biotin-mediated pull-down to generate high-complexity libraries. We further outline a computational workflow that extends beyond standard topological mapping of compartments and topologically associating domains to identify cancer-specific aberrations. Specifically, we focus on detecting chromosomal rearrangements (structural variants) and characterizing the distinct circular topology of extrachromosomal DNA. This integrated experimental and analytical framework provides the necessary tools to dissect the spatial dysregulation underlying tumor evolution.

Humans↗

PubMed2027

Teratoma Formation and Genomic Profiling Using Multi-Omics Approaches.

Teratoma formation is the gold standard assay for evaluating the developmental pluripotency of human and mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Following subcutaneous injection into immunodeficient mice, pluripotent stem cells spontaneously differentiate into derivatives representing all three embryonic germ layers-ectoderm, mesoderm, and endoderm. Beyond serving as a functional assay for pluripotency, teratomas provide a unique three-dimensional model system for studying early human development and lineage specification in vivo. This chapter describes comprehensive protocols for teratoma formation in immunodeficient mice, tissue processing for multiple downstream genomic applications, and multi-omics profiling approaches. We detail methods for embryonic stem cell culture, teratoma generation via subcutaneous injection, tissue dissection and processing for chromatin immunoprecipitation followed by sequencing (ChIP-Seq), RNA sequencing (RNA-Seq), single-cell multiome profiling combining chromatin accessibility (ATAC-Seq) and gene expression (scRNA-Seq), and histological analysis using hematoxylin and eosin (H&E) staining. Additionally, we provide bioinformatics workflows for analyzing the resulting genomic datasets to characterize the epigenetic and transcriptional landscapes of teratoma-derived tissues. These methods enable comprehensive molecular characterization of developmental processes and provide valuable resources for stem cell biologists studying pluripotency, differentiation, and early embryonic development.

Teratoma↗

PubMed2004

Functional neuronavigation and intraoperative MRI.

Our concept of computer assisted surgery is based on the combination of intraoperative magnetic resonance (MR) imaging with microscope-based neuronavigation, providing anatomical and functional guidance simultaneously. Intraoperative imaging evaluates the extent of a resection, while the additional use of functional neuronavigation, which displays the position of eloquent brain areas in the operative field, prevents increasing neurological deficits, which would otherwise result from extended resections. Up to mid 2001 we performed intraoperative MR imaging using a low-field 0.2 Tesla scanner in 330 patients. The main indications were the evaluation of the extent of resection in gliomas, pituitary tumours, and in epilepsy surgery. Intraoperative MR imaging proved to serve as intraoperative quality control with the possibility of an immediate modification of the surgical strategy, i.e. extension of the resection. Integrated use of functional neuronavigation prevented increased neurological deficits. Compared to routine pre- or postoperative imaging being performed with high-Tesla machines, intraoperative image quality and sequence spectrum could not compete. This led to the development of the concept to adapt a high-field MR scanner to the operating environment, preserving the benefits of using standard microsurgical equipment and microscope-based neuronavigational guidance with integrated functional data, which was successfully implemented by April 2002. Up to the end of 2002, 95 patients were investigated with the new setup. Improved image quality, intraoperative workflow, as well as enhanced sophisticated intraoperative imaging possibilities are the major benefits of the high-field setup.

Brain Neoplasms↗

PubMed2001

A complication conference for internal quality control at the Neurosurgical Department of the University of Heidelberg.

The registration of adverse events is an important issue in the field of medicine. Even today adverse event screening and registration is not part of the routine in most medical areas. In 1994, the Department of Neurosurgery at the University of Heidelberg implemented a conference for screening and registering adverse events. The aim was to record all complications occurring for an internal quality control. High priority was given to improving the process of data screening and registering. The conference is held every 2 weeks and all medical staff and residents of the department are obligated to be present. Screening of the adverse events encompasses all operations performed during a bi-weekly period. Every single operation is revised for an adverse event during or following the hospital stay. Adverse events are registered on a standardized data sheet and later transferred to a database for use in further investigations. After 6 years, the conference has been fully accepted and become an integral part of the workflow of the department. During this period, 8160 operations were screened and 1335 adverse events registered. The next step will be to integrate the data-collection process into the daily ward rounds using a personal digital assistant (PDA). This process is less time consuming and may perhaps augment the number of registered cases.

Germany↗

PubMed2000

Relevant priors prefetching algorithm performance for a picture archiving and communication system.

Proper prefetching of relevant prior examinations from a picture archiving and communication system (PACS) archive, when a patient is scheduled for a new imaging study, and sending the historic images to the display station where the new examination is expected to be routed and subsequently read out, can greatly facilitate interpretation and review, as well as enhance radiology departmental workflow and PACS performance. In practice, it has proven extremely difficult to implement an automatic prefetch as successful as the experienced fileroom clerk. An algorithm based on defined metagroup categories for examination type mnemonics has been designed and implemented as one possible solution to the prefetch problem. The metagroups such as gastrointestinal (GI) tract, abdomen, chest, etc, can represent, in a small number of categories, the several hundreds of examination types performed by a typical radiology department. These metagroups can be defined in a table of examination mnemonics that maps a particular mnemonic to a metagroup or groups, and vice versa. This table is used to effect the prefetch rules of relevance. A given examination may relate to several prefetch categories, and preferences are easily configurable for a particular site. The prefetch algorithm metatable was implemented in database structured query language (SQL) using a many-to-many fetch category strategy. Algorithm performance was measured by analyzing the appropriateness of the priors fetched based on the examination type of the current study. Fetched relevant priors, missed relevant priors, fetched priors that were not relevant to the current examination, and priors not fetched that were not relevant were used to calculate sensitivity and specificity for the prefetch method. The time required for real-time requesting of priors not previously prefetched was also measured. The sensitivity of the prefetch algorithm was determined to be 98.3% and the specificity 100%. Time required for on-demand requesting of priors was 9.5 minutes on average, although this time varied based on age of the prior examination and on the time of day and database traffic. A prefetch algorithm based on metatable examination mnemonic categories can pull the most appropriate relevant priors, reduce the number of missed relevant priors, and therefore reduce the time involved for the manual task of on-demand requests of priors. Network and database traffic can be reduced as well by decreasing the number of priors selected from the archive and subsequently transmitted to the display stations, through elimination of transactions on examinations not relevant to the current study.

Algorithms↗

PubMed2000

Adoption of alternative financing strategies to increase the diffusion of picture archiving and communication systems into the radiology marketplace.

The objective of the study was to evaluate current marketplace conditions and strategies employed by major picture archiving and communication systems (PACS) vendors in the creation of alternative financing strategies, to enhance the diffusion of filmless imaging. Data were collected from the major PACS vendors in the forms of survey questionnaires and review of existing leases. Topics evaluated in the survey included current financing options available, foreseeable changes in PACS financing, role of third-party financiers, and creation of risk-sharing arrangements. Generic leases were also reviewed evaluating the presence or absence of several key variables including technology obsolescence protection, hardware/software upgrades, end-of-term options, determination of fair market value, functionality/acceptance testing, uptime guarantees, and workflow management consulting. Eight of the 10 PACS vendors surveyed participated in the data collection. The vast majority of current PACS implementations (60% to 90%) occur through direct purchase, with conventional leasing (operating or capital) accounting for only 5% to 30% of PACS installations. The majority of respondents view fee-for-lease arrangements and other forms of risk sharing as increasing importance for future PACS financing. The specific targets for such risk-sharing arrangements consist of small hospital and privately owned imaging centers. Leases currently offered range in duration from 3 to 5 years and frequently offer technology obsolescence protection with upgrades, multiple end-of-term options, and some form of acceptance testing. A number of important variables frequently omitted from leases include uptime guarantees, flexibility in changing financing or vendors, and incorporation of expected productivity/operational efficiency gains. As vendors strive to increase the penetration of PACS into the radiology marketplace, there will be a shift from conventional financing (loan or purchase) to leasing. Fee-for-use leasing and other forms of risk sharing have the greatest potential in smaller hospitals, which do not have the financial resources to pursue conventional financing options. Potential PACS customers must be cautious when entering into these alternative financing strategies, to ensure that appropriate safeguards are incorporated, in order to minimize downside risk.

Cost Sharing↗

PubMed2000

Using technology assessment as the picture archiving and communication system spreads outside radiology to the enterprise.

Picture archiving and communication systems (PACS) are being implemented within radiology departments, and many facilities are entering the next stage of PACS use by deploying PACS to departments outside of radiology and to other facilities located at a distance. Many PACS vendors and department administrators have based cost-justification analyses on the anticipated savings from expanding PACS to these areas. However, many of these cost-savings analyses can be highly suspect in their assumptions and findings. Technology assessment (TA) at the hospital/health system level is an organized, systematic approach to examining the efficacy of a technology in relation to the health system's mission and clinical needs. It can be an organized and unifying approach to aid in the distribution of limited capital resources. As extra-radiology PACS deployment is a costly endeavor, TA may be used to plan for PACS implementation throughout the enterprise. In many organizations, PACS is thought of as a radiology domain as its first uses were centered on this image-producing service. Now, as PACS technology spreads to other service areas, such as cardiology, dermatology, pathology, orthopedics, obstetrics, etc, the need to incorporate other viewpoints in a system-based PACS is necessary to avoid having independent PACS that may duplicate archives and may not communicate with each other. How to meet the diverse PACS needs of clinical services can be a challenging task; a TA program has been demonstrated to effectively handle the clinical needs, demands, and timeframes of PACS planning and support throughout hospitals and health systems. A hospital-based TA program can assist health care organizations to present PACS as a system-wide need and program rather than a radiology-based program gobbling up the capital budget. Submitting PACS to the TA review process can identify essential elements in planning and help avoid many of the pitfalls of PACS implementation and operations. Thorough cost and/or return on investment analyses, phasing decisions, workflow re-engineering, and outcomes assessment programs are a few of the issues that a TA program can address to help in the transition to a complete electronic image environment. The TA process includes clinician selection, evaluation criteria and their selection for technologies under review, a policy for review/authorization/denial, and measurement of expected outcomes.

Cost Savings↗