3C Method Research Articles

The Clinical Validation of a Common Analytical Change Criteria for Cardiac Troponin for Ruling in an Acute Cardiovascular Outcome in Patients Presenting with Ischemic Chest Pain Symptoms.

Serial cardiac troponin (cTn) testing on patients with symptoms suggestive of acute coronary syndrome (ACS) is primarily to identify those patients with evolving myocardial injury. With the improved analytical performance of the high-sensitivity cTn (hs-cTn) assays, different change criteria have been proposed that are mostly assay dependent. Here, we developed and compared a new Common Change Criteria (3C for the combined criteria of >3 ng/L, >30%, or >15% based on the initial cTn concentration of <10 ng/L, 10 to 100 ng/L, or >100 ng/L, respectively) method, versus the 2 h assay-dependent absolute change criteria endorsed by the European Society of Cardiology (ESC), versus the common relative >20% change criterion. These different analytical change criteria were evaluated in 855 emergency department (ED) patients with symptoms of ACS and who had two samples collected 3 h apart. The cTn concentrations were measured with four different assays (Abbott hs-cTnI, Roche hs-cTnT, Ortho cTnI-ES, and Ortho hs-cTnI). The outcomes evaluated were myocardial infarction (MI) and a composite outcome (MI, unstable angina, ventricular arrhythmia, heart failure, or cardiovascular death) within 7 days of ED presentation. The combined change criteria (3C) method yielded higher specificities (range: 93.9 to 97.2%) as compared to the >20% criterion (range: 42.3 to 88.1%) for all four assays for MI. The 3C method only yielded a higher specificity estimate for MI for the cTnI-ES assay (95.9%) versus the absolute change criteria (71.7%). Similar estimates were obtained for the composite outcome. There was also substantial agreement between hs-cTnT and the different cTnI assays for MI with the 3C method, with the percent agreement being ≥95%. The Common Change Criteria (3C) method combining both absolute and different percent changes may be used with cTnI, hs-cTnT, and different hs-cTnI assays to yield similar high-specificity (rule-in) estimates for adverse cardiovascular events for patients presenting to the ED with ACS symptoms.

Determining chromatin architecture with Micro Capture-C.

Micro Capture-C (MCC) is a chromatin conformation capture (3C) method for visualizing reproducible three-dimensional contacts of specified regions of the genome at base pair resolution. These methods are an established family of techniques that use proximity ligation to assay the topology of chromatin. MCC can generate data at substantially higher resolution than previous techniques through multiple refinements of the 3C method. Using a sequence agnostic nuclease, the maintenance of cellular integrity and full sequencing of the ligation junctions, MCC achieves subnucleosomal levels of resolution, which can be used to reveal transcription factor binding sites analogous to DNAse I footprinting. Gene dense regions, close-range enhancer-promoter contacts, individual enhancers within super-enhancers and multiple other types of loci or regulatory regions that were previously challenging to assay with conventional 3C techniques, are readily observed using MCC. MCC requires training in common molecular biology techniques and bioinformatics to perform the experiment and analyze the data. The protocol can be expected to be completed in a 3 week timeframe for experienced molecular biologists.

Application of the 3C Method to Study the Developmental Genes in Drosophila Larvae.

A transition from one developmental stage to another is accompanied by activation of developmental programs and corresponding gene ensembles. Changes in the spatial conformation of the corresponding loci are associated with this activation and can be investigated with the help of the Chromosome Conformation Capture (3C) methodology. Application of 3C to specific developmental stages is a sophisticated task. Here, we describe the use of the 3C method to study the spatial organization of developmental loci in Drosophila larvae. We critically analyzed the existing protocols and offered our own solutions and the optimized protocol to overcome limitations. To demonstrate the efficiency of our procedure, we studied the spatial organization of the developmental locus Dad in 3rd instar Drosophila larvae. Differences in locus conformation were found between embryonic cells and living wild-type larvae. We also observed the establishment of novel regulatory interactions in the presence of an adjacent transgene upon activation of its expression in larvae. Our work fills the gap in the application of the 3C method to Drosophila larvae and provides a useful guide for establishing 3C on an animal model.

Subtelomeric Chromatin Structure by Chromosome Conformation Capture (3C)-qPCR Methodology in Candida glabrata.

Chromatin architecture has an enormous impact on gene regulation, DNA replication, repair, and packaging. Chromatin is organized in a complex hierarchical manner in which distant fragments of DNA can interact with each other through DNA loops. DNA loops can interact between themselves to form topologically associated domains (TADs) that are further organized into functional compartments. In the last two decades, Chromatin Conformation Capture (3C technology) and its high-throughput derivatives allowed detailed analysis of the chromatin architecture. The 3C method is based on ligation of distant fragments brought together by DNA looping. The method analyzes a particular genomic region of interest and quantifies the interactions between a defined fragment with all the surrounding fragments of the region. It consists of four steps: (1) The long-distance interacting chromatin fragments are fixed with formaldehyde in whole cells which are then lysed; (2) the fixed chromatin is digested with a carefully chosen restriction enzymes to separate intervening DNA fragments; (3) the fragments brought into proximity by DNA looping are ligated in conditions favoring intramolecular ligation; and (4) the interactions are quantified by quantitative PCR using the TaqMan technology and unidirectional primers. Herein, we describe the use of this methodology to analyze the chromatin conformation at a subtelomeric locus containing three genes encoding adhesins and several cis-regulatory elements, in the pathogenic yeast Candida glabrata.

Quantitative Chromosome Conformation Capture (3C-qPCR).

Many population-based methods investigating chromatin dynamics and organization in eukaryotes are based on the chromosome conformation capture (3C) method. Here, we provide an updated version of the quantitative 3C (3C-qPCR) protocol for improved and simplified quantitative analyses of intra-chromosomal contacts.

Rancangan Strategi Media Sosial PT XYZ pada Tahun 2021

PT XYZ is a low-cost airline or low-cost airline, which is actively engaged in developing the company's digital presence to increase transaction rates and assess relationships with relevant and trusted consumers. Recently, the aviation industry has faced tough challenges, especially the Covid-19 pandemic that has hit the world. Indonesian airline companies have long been active in building their digital presence to become market leaders, these airlines can use social media; Instagram, Facebook and Twitter. This research hopes to help PT XYZ increase its competitive advantage by analyzing the 3C method, namely Customer, Competitor, and Company analysis. The data obtained for the research is the result of survey data from apprenticeship reports, internet observations (netnography), and study report documents. With the data that has been obtained, a SWOT analysis is then carried out to find weaknesses, opportunities, and threats that the company could have. The results are then used to plan with the TOWS Matrix to produce several social media designs that can be implemented by PT XYZ. The resulting strategic design to increase corporate transactions are; (1) photo contest, (2) story contest, (3) open channels on Whatsapp social media (4) use Instagram Shops, and (5) collaborate with content creators on the Youtube platform. The strategic design undertaken is expected to increase consumer transactions in the B2C market.

From Hi-C Contact Map to Three-Dimensional Organization of Interphase Human Chromosomes

The probability of two loci, separated by a certain genome length, being in contact can be inferred using the Chromosome Conformation Capture (3C) method and related Hi-C experiments. How to go from the contact map, a matrix listing the mean contact probabilities between a large number of pairs of loci, to an ensemble of three-dimensional structures is an open problem. A solution to this problem, without assuming an assumed energy function, would be the first step in understanding the way nature has solved the packaging of chromosomes in tight cellular spaces. We created a theory, based on polymer physics characteristics of chromosomes and the maximum entropy principles, referred to as HIPPS (Hi-C-Polymer-Physics-Structures) method, that allows us to calculate the 3D structures solely from Hi-C contact maps. We created an ensemble of 3D structures for the 23 chromosomes from lymphoblastoid cells using the measured contact maps as inputs. The HIPPS method shows that conformations of chromosomes are heterogeneous even in a single cell type. The differences in the conformational heterogeneity of the same chromosome in different cell types (normal as well as cancerous cells) can also be quantitatively discerned using our theory. We validate the method by showing that the calculated volumes of the 23 chromosomes from the predicted 3D structures are in good agreement with experimental estimates. Because the method is general, the 3D structures for any species may be calculated directly from the contact map without the need to assume a specific polymer model, as is customarily done.

Context, class, and community: a methodological framework for studying labor organizing and digital unionizing

ABSTRACT A key debate in an increasingly digital environment is the tension between individuals and institutions with online engagement. An area that exemplifies this opposition is the study of digital unionizing. Labor studies scholarship tends to focus on institutions while communication research often privileges the individual. This article extends these two approaches by outlining three key considerations in what shapes online collective action within working-class and worker struggles: context, class and community. I also conceptualize this methodological framework to study digital unionizing and labor organizing more broadly, which I refer to as the 3C Method. The empirical foundation for this methodological argument derives from existing literature, as well as select findings from a three-year digital organizing research project.

High-resolution targeted 3C interrogation of cis-regulatory element organization at genome-wide scale

Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods generally provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at limited numbers of loci. Due to technical limitations, generation of reproducible high-resolution interaction profiles has not been achieved at genome-wide scale. Here, to overcome this barrier, we systematically test each step of 3C and report two improvements over current methods. We show that up to 30% of reporter events generated using the popular in situ 3C method arise from ligations between two individual nuclei, but this noise can be almost entirely eliminated by isolating intact nuclei after ligation. Using Nuclear-Titrated Capture-C, we generate reproducible high-resolution genome-wide 3C interaction profiles by targeting 8055 gene promoters in erythroid cells. By pairing high-resolution 3C interaction calls with nascent gene expression we interrogate the role of promoter hubs and super-enhancers in gene regulation.

Enhanced Circular Chain Control for Parallel Operation of Inverters in UPS Systems

In this paper, a current sharing method based on the circular chain control (3C) method is proposed for controlling parallel inverters of unequal ratings in uninterruptible power supply (UPS) applications. Due to its circular structure, 3C is one of the most convenient methods which can be used in UPS as well as microgrid systems. However, the conventional 3C control strategy is only applicable to inverters of equal power ratings. The proposed method not only retains the circular structure of the 3C method, but also provides adaptability for the parallel operation of inverters with different power ratings. Moreover, this method adds hot-swap capability to the parallel inverter. A two-loop control structure is used to control the inverters. For proper current sharing, currents of inverters are conveyed in a circular structure with appropriate gains through control links. Simulation and experimental results for linear and nonlinear loads verify the effectiveness of the proposed strategy.

4C-seq from beginning to end: A detailed protocol for sample preparation and data analysis.

Chromosome conformation capture (3C) methods measure DNA contact frequencies based on nuclear proximity ligation, to uncover in vivo genomic folding patterns. 4C-seq is a derivative 3C method, designed to search the genome for sequences contacting a selected genomic site of interest. 4C-seq employs inverse PCR and next generation sequencing to amplify, identify and quantify its proximity ligated DNA fragments. It generates high-resolution contact profiles for selected genomic sites based on limited amounts of sequencing reads. 4C-seq can be used to study multiple aspects of genome organization. It primarily serves to identify specific long-range DNA contacts between individual regulatory DNA modules, forming for example regulatory chromatin loops between enhancers and promoters, or architectural chromatin loops between cohesin- and CTCF- associated domain boundaries. Additionally, 4C-seq contact profiles can reveal the contours of contact domains and can identify the structural domains that co-occupy the same nuclear compartment. Here, we present an improved step-by-step protocol for sample preparation and the generation of 4C-seq sequencing libraries, including an optimized PCR and 4C template purification strategy. In addition, a data processing pipeline is provided which processes multiplexed 4C-seq reads directly from FASTQ files and generates files compatible with standard genome browsers for visualization and further statistical analysis of the data such as peak calling using peakC. The protocols and the pipeline presented should readily allow anyone to generate, visualize and interpret their own high resolution 4C contact datasets.

Diploid genome in 3D.

Dip-C, a descendent of the 3C method, reveals 3D genome structures of single diploid human cells.

Microfluidics-Based Chromosome Conformation Capture (3C) Technology for Examining Chromatin Organization with a Low Quantity of Cells.

Detecting three-dimensional (3D) genome organization in the form of physical interactions between various genomic loci is of great importance for understanding transcriptional regulations and cellular fate. Chromosome Conformation Capture (3C) method is the gold standard for examining chromatin organization, but usually requires a large number of cells (>107). This hinders studies of scarce tissue samples from animals and patients using the method. Here we developed a microfluidics-based approach for examining chromosome conformation by 3C technology. Critical 3C steps, such as digestion and religation of BAC DNA and cross-linked chromatin, were implemented on a microfluidic chip using a low quantity of cells (<104). Using this technology, we analyzed the chromatin looping interactions in the human β-globin. We envision that our method will provide a powerful tool for low-input analysis of chromosome conformation and epigenetic regulations.

Practical Analysis of Hi-C Data: Generating A/B Compartment Profiles.

Recent advances in next-generation sequencing (NGS) and chromosome conformation capture (3C) analysis have led to the development of Hi-C, a genome-wide version of the 3C method. Hi-C has identified new levels of chromosome organization such as A/B compartments, topologically associating domains (TADs) as well as large megadomains on the inactive X chromosome, while allowing the identification of chromatin loops at the genome scale. Despite its powerfulness, Hi-C data analysis is much more involved compared to conventional NGS applications such as RNA-seq or ChIP-seq and requires many more steps. This presents a significant hurdle for those who wish to implement Hi-C technology into their laboratory. On the other hand, genomics data repository sites sometimes contain processed Hi-C data sets, allowing researchers to perform further analysis without the need for high-spec workstations and servers. In this chapter, we provide a detailed description on how to calculate A/B compartment profiles from processed Hi-C data on the autosomes and the active/inactive X chromosomes.

Long-distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region upregulating the allele-specific MYC expression in HeLa cells.

Human papillomavirus (HPV) infection is the most important risk factor for cervical cancer development. In HeLa cell line, the HPV viral genome is integrated at 8q24 in one allele of chromosome 8. It has been reported that the HPV fragment integrated in HeLa genome can cis‐activate the expression of proto‐oncogene MYC, which is located at 500 kb downstream of the integrated site. However, the underlying molecular mechanism of this regulation is unknown. A recent study reported that MYC was highly expressed exclusively from the HPV‐integrated haplotype, and a long‐range chromatin interaction between the integrated HPV fragment and MYC gene has been hypothesized. In this study, we provided the experimental evidences supporting this long‐range chromatin interaction in HeLa cells by using Chromosome Conformation Capture (3C) method. We found that the integrated HPV fragment, MYC and 8q24.22 was close to each other and might form a trimer in spatial location. When knocking out the integrated HPV fragment or 8q24.22 region from chromosome 8 by CRISPR/Cas9 system, the expression of MYC reduced dramatically in HeLa cells. Interestingly, decreased expression was only observed in three from eight cell clones, when only one 8q24.22 allele was knocked out. Functionally, HPV knockout caused senescence‐associated acidic β‐gal activity in HeLa cells. These data indicate a long‐distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region, providing an alternative mechanism relevant to the carcinogenicity of HPV integration.

Genome-wide analysis of chromatin packing in Arabidopsis thaliana at single-gene resolution.

The three-dimensional packing of the genome plays an important role in regulating gene expression. We have used Hi-C, a genome-wide chromatin conformation capture (3C) method, to analyze Arabidopsis thaliana chromosomes dissected into subkilobase segments, which is required for gene-level resolution in this species with a gene-dense genome. We found that the repressive H3K27me3 histone mark is overrepresented in the promoter regions of genes that are in conformational linkage over long distances. In line with the globally dispersed distribution of RNA polymerase II in A. thaliana nuclear space, actively transcribed genes do not show a strong tendency to associate with each other. In general, there are often contacts between 5′ and 3′ ends of genes, forming local chromatin loops. Such self-loop structures of genes are more likely to occur in more highly expressed genes, although they can also be found in silent genes. Silent genes with local chromatin loops are highly enriched for the histone variant H3.3 at their 5′ and 3′ ends but depleted of repressive marks such as heterochromatic histone modifications and DNA methylation in flanking regions. Our results suggest that, different from animals, a major theme of genome folding in A. thaliana is the formation of structural units that correspond to gene bodies.

Chromosome Conformation Capture in Primary Human Cells.

3D organization of the genome, its structural and regulatory function of cell identity, is acquiring prominent features in epigenetics studies; more efforts have been done to develop techniques that allow studying nuclear structure. Chromosome conformation capture (3C) has been set up in 2002 from Dekker and from that moment great investments were made to develop genomics variants of 3C technology (4C, 5C, Hi-C) providing new tools to investigate the shape of the genome in a more systematic and unbiased manner. 3C method allows scientists to fix dynamic and variable 3D interactions in nuclear space, and consequently to study which sequences interact, how a gene is regulated by different and distant enhancer, or how a set of enhancer could regulate transcriptional units; to follow the conformation that mediates regulation change in development; and to evaluate if this fine epigenetic mechanism is impaired in disease condition.

Detecting Spatial Chromatin Organization by Chromosome Conformation Capture II: Genome-Wide Profiling by Hi-C.

The chromosome conformation capture (3C) method has been invaluable in studying chromatin interactions in a population of cells at a resolution surpassing that of light microscopy, for example in the detection of functional contacts between enhancers and promoters. Recent developments in sequencing-based chromosomal contact mapping (Hi-C, 5C and 4C-Seq) have allowed researchers to interrogate pairwise chromatin interactions on a wider scale, shedding light on the three-dimensional organization of chromosomes. These methods present significant technical and bioinformatic challenges to consider at the start of the project. Here, we describe two alternative methods for Hi-C, depending on the size of the genome, and discuss the major computational approaches to convert the raw sequencing data into meaningful models of how genomes are organized.

Chromosome Conformation Capture (3C) in Budding Yeast.

Chromosome conformation capture (3C) is a method for studying chromosomal organization that takes advantage of formaldehyde cross-linking to measure the spatial association of two pieces of chromatin. The 3C method begins with whole-cell formaldehyde fixation of chromatin. After cell lysis, solubilized chromatin is digested with a type II restriction endonuclease, and cross-linked DNA fragments are ligated together. Cross-links are reversed by degradation with proteinase K, and chimeric DNA molecules are purified by standard phenol:chloroform extraction. The resulting 3C library represents chromatin fragments that may be separated by large genomic distances or located on different chromosomes, but are close enough in three-dimensional space for cross-linking. Locus-specific oligonucleotide primers are used to detect interactions of interest in the 3C library using end-point polymerase chain reaction (PCR).

Chromosome conformation capture (3C) of tandem arrays in yeast.

Studying interphase chromosome arrangements at the molecular level can provide important details on the function and coordination of many metabolic processes that take place on DNA, such as transcription or DNA repair. The chromosome conformation capture (3C) methodology was originally developed in yeast to study the interphase organization of a single-yeast chromosome. 3C assays allow the identification of physical interactions between distant DNA segments and thus provide detailed information on the folding of chromatin in the native cellular state. Since its initial development, the technique has been used to advance our understanding of the folding of gene loci and has yielded important discoveries, like the demonstration that distant transcriptional regulatory elements interact with their target promoters through chromatin loops. The 3C method uses formaldehyde cross-linking to covalently link interacting chromatin segments in intact cells. After chromatin digestion and ligation of cross-linked fragments, the frequency of interaction between distant DNA elements can be quantified. However, the design, analysis, and controls used are critical when using 3C. In this chapter, we describe the general protocol for 3C analysis and demonstrate it through analysis of interactions in repetitive sequences of the ribosomal gene array of Saccharomyces cerevisiae.