Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

Metastasis occurs when cancer cells leave the primary tumour and travel to a secondary site to form a new lesion. The tumour microenvironment (TME) is recognised to greatly influence this process, with for instance the vascular system enabling the dissemination of the cells into other tissues. However, understanding the exact role of these microenvironmental cells during metastasis has proven challenging. Indeed, in vitro models often appear too simplistic, and the study of the interactions between different cell types in a 3D space is limited. On the other hand, even though in vivo models incorporate the TME, observing cells in real-time to understand their exact role is difficult. Horizontal compartmentalised microfluidic models are a promising new platform for metastasis studies. These devices, composed of adjacent microchannels, can incorporate multiple cell types within a 3D space. Furthermore, the transparency and thickness of these models also enables high quality real-time imaging to be performed. This paper demonstrates how these devices can be successfully used for oral squamous cell carcinoma (OSCC) metastasis studies, focusing on the role of the vascular system in this process. Conditions for co-culture of OSCC cells and endothelial cells have been determined and staining protocols optimised. Furthermore, several imaging analysis techniques for these models are described, enabling precise segmentation of the different cell types on the images as well as accurate assessment of their phenotype. These methods can be applied to any study aiming to understand the role of microenvironmental cell types in cancer metastatic dissemination, and overcome several challenges encountered with current in vitro and in vivo models. Hence, this new in vitro model capable of recapitulating important aspects of the cellular complexity of human metastatic dissemination can ultimately contribute to replacing animal studies in this field.

In plants, gene expression is orchestrated by thousands of transcription factors (TFs). For instance, a large set of bHLH TFs are involved in the regulation of iron homeostasis in Arabidopsis thaliana. The identification of the direct target genes of TFs through uncovering the interaction between the TFs and cis-regulatory elements has become an essential step toward a comprehensive understanding of the iron homeostasis transcriptional regulatory network in Arabidopsis. Chromatin immunoprecipitation (ChIP) followed by qRT-PCR (ChIP-qPCR), sequencing (ChIP-seq), or chip hybridization (ChIP-chip) is a robust tool to investigate protein-DNA interactions in plants in a physiological context. The procedure generally includes six steps: DNA-protein crosslink, isolation of nuclei, shearing of chromatin, immunoprecipitation, DNA purification, and qRT-PCR analyses. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP experiment in Arabidopsis. This protocol focuses on seedlings grown in control and iron deficiency conditions, but can readily be adapted for use with other Arabidopsis tissues or samples. In addition, the protocol could also be applied to perform ChIP-chip or ChIP-seq experiments.

Cardiac allograft vasculopathy (CAV) is a leading cause of late graft failure and mortality after heart transplantation (HT). Sharing some features with atherosclerosis, CAV results in diffuse narrowing of the epicardial coronaries and microvasculature, with consequent graft ischemia. Recently, clonal hematopoiesis of indeterminate potential (CHIP) has emerged as a risk factor for cardiovascular disease and mortality. We aimed to investigate the relationship between CHIP and posttransplant outcomes, including CAV. We analyzed 479 HT recipients with stored DNA samples at 2 high-volume transplant centers, Vanderbilt University Medical Center and Columbia University Irving Medical Center. We explored the association between the presence of CHIP mutations with CAV and mortality after HT. In this case-control analysis, carriers of CHIP mutations were not at increased risk of CAV or mortality after HT. In a large multicenter genomics study of the heart transplant population, the presence of CHIP mutations was not associated with an increased risk of CAV or posttransplant mortality.

The epigenome consists of all the epigenetic alterations like DNA methylation, the histone modifications and non-coding RNAs which change the gene expression and have a role in diseases like cancer and other processes. Epigenetic modifications can control gene expression through variable gene activity at various levels which affects various cellular phenomenon such as cell differentiations, variability, morphogenesis, and the adaptability of an organism. Various factors such as food, pollutants, drugs, stress etc., impact the epigenome. Epigenetic mechanisms mainly involve various post-translational alteration of histones and DNA methylation. Numerous methods have been utilized to study these epigenetic marks. Various histone modifications and binding of histone modifier proteins can be analyzed using chromatin immunoprecipitation (ChIP) which is one of broadly utilized method. Other modified forms of the ChIP have been developed such as reverse chromatin immunoprecipitation (R-ChIP); sequential ChIP (ChIP-re-ChIP) and some high-throughput modified forms of ChIP such as ChIP-seq and ChIP-on-chip. Another epigenetic mechanism is DNA methylation, in which DNA methyltransferases (DNMTs) add a methyl group to the C-5 position of the cytosine. Bisulfite sequencing is the oldest and usually utilized method to measure the DNA methylation status. Other techniques have been established are whole genome bisulfite sequencing (WGBS), methylated DNA immune-precipitation based methods (MeDIP), methylation sensitive restriction enzyme digestion followed by sequencing (MRE-seq) and methylation BeadChip to study the methylome. This chapter briefly discusses the key principles and methods used to study epigenetics in health and disease conditions.

UNASSIGNED: IDH1/2 mutations, intervening in epigenetic procedures, are frequently encountered in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Knowledge of the genetics, immunophenotypes, and mutational kinetics of IDH1/2-mutated AML can contribute to the understanding of AML clonal architecture and inform therapeutics and monitoring.
UNASSIGNED: We retrospectively analyzed 50 IDH1/2-mutated AML/MDS-EB cases of our institution, to identify recurrent co-mutations, immunophenotypes, patterns of co-variance of IDH1/2 allele burdens with those of recurrent co-mutations, frequency of persistent IDH1/2 mutation as clonal hematopoiesis of indeterminate potential (CHIP) in remission and response to hypomethylating agents.
UNASSIGNED: Most frequently co-mutated genes were DNMT3A, SRSF2 and NPM1. Most cases with co-existent IDH1/2 and NPM1 mutations (11/13) showed an \'APL-like\' immunophenotype (CD34-HLADR-). Allele burdens of mutated IDH1/2 were identical to mutated SRSF2 allele burdens at diagnosis and remission, but not always to mutated NPM1 allele burden in remission. We show persistence of significant mutIDH1/2 allele burden in approximately one-fourth of patients with deep remissions. IDH1/2 mutations were significantly more frequent among responders to first-line HMA-based regimens than among non-responders, in patients treated for myeloid neoplasms with excess blasts.
UNASSIGNED: IDH1/2 mutations are most frequently accompanied by DNMT3A, SRSF2 and NPM1 mutations. NPM1-IDH1/2 mutated AML has a mature phenotype possibly amenable to differentiation therapies. IDH1/2 and SRSF2 mutations probably arise at the same developmental stage of the disease, as their allele burdens covariate. IDH1/2 mutation represents CHIP in a substantial proportion of cases and is therefore no reliable residual disease marker. The preferential presence of IDH1/2 mutations among HMA-responders could inform therapeutic decisions if confirmed in larger series.

UNASSIGNED: Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method using only one type of enzyme that can amplify DNA with high specificity, efficiency and rapidity under isothermal conditions. Chips for Complicated Infection Detection (CCID) is based on LAMP. This study translate CCID into clinical application and evaluate its diagnostic value for pneumonia.
UNASSIGNED: Eighty one older patients with pneumonia were prospectively enrolled from January 1 to July 23, 2021, and 57 sputum/airway secretion and 35 bronchoalveolar lavage fluid samples were collected and analyzed by CCID and conventional microbiological tests (CMTs). Samples were collected, transported, monitored, and managed by a multidisciplinary team using a sample management information system.
UNASSIGNED: CCID turnaround time was 50 min, and the detection limit was 500 copies/reaction. The percentage of positive samples was significantly higher using CCID than CMTs, especially for Klebsiella pneumoniae (odds ratio [OR], 9.0; 95% confidence interval [CI], 1.1-70.5; p < 0.05), Enterococcus faecalis (OR, ∞; p < 0.01), Stenotrophomonas maltophilia (OR, ∞; p < 0.01), fungi (OR, 26.0; 95% CI, 3.6-190.0; p < 0.01), and viruses (CCID only; p < 0.01). In addition, the percentage of positive results was significantly higher using CCID than CMTs in patients who used antibiotics for more than 3 days (91.9% vs. 64.9%; p < 0.01). Analyzing clinical impact, 55 cases (59.8%) benefited from CCID.
UNASSIGNED: CCID allows the rapid and accurate detection of pneumonia in older patients. Moreover, this technique is less affected by previous antibiotic treatment and can improve patient care.

暂无摘要。

The myeloproliferative neoplasms are associated with chronic kidney disease but whether clonal haematopoiesis of indeterminate potential (CHIP) is associated with impaired kidney function is unknown. In the Danish General Suburban Population Study (N = 19 958) from 2010 to 2013, 645 individuals were positive for JAK2V617F (N = 613) or CALR (N = 32) mutations. Mutation-positive individuals without haematological malignancy were defined as having CHIP (N = 629). We used multiple and inverse probability weighted (IPW)-adjusted linear regression analysis to estimate adjusted mean (95% confidence interval) differences in estimated glomerular filtration rate (eGFR; ml/min/1.73 m2 ) by mutation status, variant allele frequency (VAF%), blood cell counts, and neutrophil-to-lymphocyte ratio (NLR). We performed 11-year longitudinal follow-up of eGFR in all individuals. Compared to CHIP-negative individuals, the mean differences in eGFR were -5.6 (-10.3, -0.8, p = .02) for CALR, -11.9 (-21.4, -2.4, p = 0.01) for CALR type 2, and -10.1 (-18.1, -2.2, p = .01) for CALR with VAF ≥ 1%. The IPW-adjusted linear regression analyses showed similar results. NLR was negatively associated with eGFR. Individuals with CALR type 2 had a worse 11-year longitudinal follow-up on eGFR compared to CHIP-negative individuals (p = .004). In conclusion, individuals with CALR mutations, especially CALR type 2, had impaired kidney function compared to CHIP-negative individuals as measured by a lower eGFR at baseline and during 11-year follow-up.

Hypoxia and inflammation are intensely connected in a functional crosstalk. Within this crosstalk, two major transcription factors take center stage: HIF and NF-κB. To investigate transcription factor function, an important aspect is its ability to bind DNA. The most appropriate method to study this property in cells is the use of chromatin immunoprecipitation followed by qPCR and/or next generation sequencing. This allows identification of potentially directly regulated genes as well as enhancer regions. Here we describe the ChIP-qPCR method in detail, including key aspects important for the success of the technique.