UNASSIGNED: Multiple sclerosis (MS) is a chronic inflammatory condition that impacts the central nervous system. It is distinguished by processes like demyelination, gliosis, neuro-axonal harm, and inflammation. The prevailing theory suggests that MS originates from an immune response directed against the body\'s own antigens within the central nervous system.
UNASSIGNED: The main aim of this research paper \"Neuroinflammation-on-a-Chip\" for studying multiple sclerosis is to enhance our comprehension of MS development, demonstrate the application of cutting-edge technology, and potentially provide valuable insights for therapeutic approaches.
UNASSIGNED: The available literature for this Narrative Review was searched on various bibliographic databases, PubMed, NCBI, and many other medical references using an individually verified, prespecified approach. Studies regarding the significance of MS and its neuroinflammatory pathogenesis in addition to the development and optimization of neuroinflammatory-on-a-chip and the advancement in innovations in this field have been reviewed in this research for a better understanding of \"Neuroinflammation-on-a-chip for multiple sclerosis\". The level of evidence of the included studies was considered as per the Centre for Evidence-Based Medicine recommendations.
UNASSIGNED: Several studies have indicated that the brain-chip model closely mimics cortical brain tissue compared to commonly used conventional cell culture methods like the Transwell culture system. Additionally, these studies have clearly demonstrated that further research using brain chips has the potential to enhance our understanding of the molecular mechanisms and roles of blood-brain barrier (BBB) transporters in both normal and disease conditions.
UNASSIGNED: Understanding neuroinflammation processes remains essential to establish new MS treatments approaches. The utilization of brain chips promises to advance our understanding of the molecular processes involving BBB transporters, both in normal and diseased states. Further research needs to be addressed in order to enhance the performance and understanding of neuroinflammation on a chip, hence aiming to provide more effective treatments for all CNS diseases.

Chromatin immunoprecipitation (ChIP) is used to analyze the targeting of a protein to a specific region of chromatin in vivo. Here, we present an instructive ChIP protocol for Arabidopsis imbibed seeds. The protocol covers all steps, from the sampling of imbibed seeds to the reverse crosslinking of immunoprecipitated protein-DNA complexes, and includes experimental tips and notes. The targeting of the protein to DNA is determined by quantitative PCR (qPCR) using reverse crosslinked DNA. The protocol can be further scaled up for ChIP-sequencing (ChIP-seq) analysis. As an example of the protocol, we include a ChIP-quantitative PCR (ChIP-qPCR) analysis demonstrating the targeting of PIF1 to the ABI5 promoter.

We fabricated a microfluidic chip (osteoblast [OB]-osteoclast [OC] chip) that could regulate the mixture amounts of OB and OC supernatants to investigate the effect of different supernatant distributions on osteogenesis or osteoclastogenesis. Computer-aided design was used to produce an OB-OC chip from polydimethylsiloxane. A pressure controller was assembled and different blends of OB and OC supernatants were correctly determined. OB and OC supernatants were placed on the upper panels of the OB-OC chip after differentiation for an in vitro evaluation. We then tested the changes in osteogenesis using MC3T3-E1 cells in the middle chambers. We observed that a 75:25 distribution of OB and OC supernatants was the most potent in osteogenesis. We then primed the osteogenic differentiation of MC3T3-E1 cells using an OB-OC mixed supernatant or an OB supernatant alone (supernatant ratios of 75:25 or 100:0, respectively). These cells were placed on the calvarial defect sites of rats. Microcomputed tomography and histological analyses determined a significantly higher bone formation in the group exposed to the OB-OC supernatant at a ratio of 75:25. In this study, we demonstrate the applicability of an OB-OC chip to evaluate the effect of different supernatant distributions of OB and OC. We observed that the highest bone-forming potential was in MC3T3-E1 cells treated with conditioned media, specifically the OB-OC supernatant at a ratio of 75:25.

In traditional cell line design pipelines, cost- and time-intensive long-term stability studies must be performed due to random integration of the transgene into the genome. By this, integration into epigenetically silenced regions can lead to silencing of the recombinant promoter over time. Site-specific integration into regions with active chromatin structure can overcome this problem and lead to strong and stable gene expression. Here, we describe a detailed protocol to identify integration sites with epigenetically preferable properties by chromatin immunoprecipitation sequencing and use them for stable and strong gene expression by applying CRISPR/Cas9. Furthermore, the examination of the integration sites with focus on Cas9-targeted sequencing with nanopores is described.

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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.

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Insulin/IGF signaling (IIS) regulates developmental and metabolic plasticity. Conditional regulation of insulin-like peptide expression and secretion promotes different phenotypes in different environments. However, IIS can also be regulated by other, less-understood mechanisms. For example, stability of the only known insulin/IGF receptor in C. elegans, DAF-2/INSR, is regulated by CHIP-dependent ubiquitination. Disruption of chn-1/CHIP reduces longevity in C. elegans by increasing DAF-2/INSR abundance and IIS activity in adults. Likewise, mutation of a ubiquitination site causes daf-2(gk390525) to display gain-of-function phenotypes in adults. However, we show that this allele displays loss-of-function phenotypes in larvae, and that its effect on IIS activity transitions from negative to positive during development. In contrast, the allele acts like a gain-of-function in larvae cultured at high temperature, inhibiting temperature-dependent dauer formation. Disruption of chn-1/CHIP causes an increase in IIS activity in starved L1 larvae, unlike daf-2(gk390525). CHN-1/CHIP ubiquitinates DAF-2/INSR at multiple sites. These results suggest that the sites that are functionally relevant to negative regulation of IIS vary in larvae and adults, at different temperatures, and in nutrient-dependent fashion, revealing additional layers of IIS regulation.

The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3\' processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3\' end-processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.

Clonal hematopoiesis of indeterminate potential (CHIP) describes recurrent somatic gene mutations in the blood of healthy individuals, associated with higher risk for hematological malignancies and higher all-cause mortality by cardiovascular disease. CHIP increases with age and is more common in adult patients after chemotherapy or radiation for cancer. Furthermore, in some adult patients undergoing autologous stem cell transplantation (ASCT) or thereafter, CHIP has been identified. In children and adolescents, it remains unclear how cellular stressors such as cytotoxic therapy influence the incidence and expansion of CHIP. We conducted a retrospective study on 33 pediatric patients mostly with solid tumors undergoing ASCT for presence of CHIP. We analyzed CD34+ selected peripheral blood stem cell grafts after several cycles of chemotherapy, prior to cell infusion, by next-generation sequencing including 18 \"CHIP-genes\". Apart from a somatic variant in TP53 in one patient no other variants indicative of CHIP were identified. As a CHIP-unrelated finding, germline variants in CHEK2 and in ATM were identified in two and four patients, respectively. In conclusion, we could not detect \"typical\" CHIP variants in our cohort of pediatric cancer patients undergoing ASCT. However, more studies with larger patient numbers are necessary to assess if chemotherapy in the pediatric setting contributes to an increased CHIP incidence and at what time point.