CTCF regulates global chromatin accessibility and transcription during rod photoreceptor development.

Chromatin architecture facilitates accurate transcription at a number of loci, but it remains unclear how much chromatin architecture is involved in global transcriptional regulation. Previous work has shown that rapid depletion of the architectural protein CTCF in cell culture strongly alters chromatin organization but results in surprisingly limited gene expression changes. This discrepancy has also been observed when other architectural proteins are depleted, and one possible explanation is that full transcriptional changes are masked by cellular heterogeneity. We tested this idea by performing multi-omics analyses with sorted post-mitotic mouse rods, which undergo synchronized development, and identified CTCF-dependent regulation of global chromatin accessibility and gene expression. Depletion of CTCF leads to dysregulation of ∼20% of the entire transcriptome (>3,000 genes) and ∼41% of genome accessibility (>26,000 sites), and these regions are strongly enriched in euchromatin. Importantly, these changes are highly enriched for CTCF occupancy, suggesting direct CTCF binding and transcriptional regulation at these active loci. CTCF mainly promotes chromatin accessibility of these direct binding targets, and a large fraction of these sites correspond to promoters. At these sites, CTCF binding frequently promotes accessibility and inhibits expression, and motifs of transcription repressors are found to be significantly enriched. Our findings provide different and often opposite conclusions from previous studies, emphasizing the need to consider cell heterogeneity and cell type specificity when performing multi-omics analyses. We conclude that the architectural protein CTCF binds chromatin and regulates global chromatin accessibility and transcription during rod development.

Potassium peroxoborate: A sustained-released reactive oxygen carrier with enhanced PAHs contaminated soil remediation performance.

Seeking for a safe, efficient, inexpensive, and eco-friendly oxidizer is always a big challenge for in-situ chemical oxidation (ISCO) technology. This study adopted the potassium peroxoborate (PPB), a novel peroxide, for soil remediation for the first time. PPB based chemical oxidation system (PPB-CO) could efficiently degrade polycyclic aromatic hydrocarbons (PAHs) without other reagents added, reaching 72.1 %, 64.2 %, and 50.0 % removal rates for naphthalene, phenanthrene, and pyrene after 24 h reaction, respectively. The superior total PAHs removal efficiency (60.6 %) was 3.6-4.7 times higher than that of other commercial peroxides (2Na2CO3•3H2O, CaO2, and H2O2). Mechanism analysis revealed that varieties of reactive oxygen species (ROS) can be generated by PPB through Fenton-like or non-Fenton routines, including H2O2, perborates species, O2•-, •OH, and 1O2. The sustainable generation of H2O2 reduced the disproportionation effect of H2O2 by 86 %, significantly improving the utilization rate. Moreover, sandbox experiments and actual contaminated soil remediation experiments verified the feasibility of PPB-CO in a real polluted site. This work provides a novel strategy for effectively soil remediation, highlighting the selection and application of new oxidants.

Boosting Efficient Alkaline Hydrogen Evolution Reaction of CoFe-Layered Double Hydroxides Nanosheets via Co-Coordination Mechanism of W-Doping and Oxygen Defect Engineering.

While surface defects and heteroatom doping exhibit promising potential in augmenting the electrocatalytic hydrogen evolution reaction (HER), their performance remains unable to rival that of the costly Pt-based catalysts. Yet, the concurrent modification of catalysts by integrating both approaches stands as a promising strategy to effectively address the aforementioned limitation. In this work, tungsten dopants are introduced into self-supported CoFe-layered double hydroxides (LDH) on nickel foam using a hydrothermal method, and oxygen vacancies (Ov) are further introduced through calcination. The analysis results demonstrated that tungsten doping reduces the Ov formation energy of CoFeW-LDH. The Ov acted as oxophilic sites, facilitating water adsorption and dissociation, and reducing the barrier for cleaving HO─H bonds from 0.64 to 0.14 eV. Additionally, Ov regulated the electronic structure of CoFeW-LDH to endow optimized hydrogen binding ability on tungsten atoms, thereby accelerating alkaline Volmer and Heyrovsky reaction kinetics. Specifically, the abundance of Ov induced a transition of tungsten from a six-coordinated to highly active four-coordinated structure, which becomes the active site for HER. Consequently, an ultra-low overpotential of 41 mV at 10 mA cm-2 , and a low Tafel slope of 35 mV dec-1 are achieved. These findings offer crucial insights for the design of efficient HER electrocatalysts.

Inhibition of RhoGEF/RhoA alleviates regorafenib resistance and cancer stemness via Hippo signaling pathway in hepatocellular carcinoma.

Patients with hepatocellular carcinoma (HCC) are vulnerable to drug resistance. Although drug resistance has been taken much attention to HCC therapy, little is known of regorafenib and regorafenib resistance (RR). This study aimed to determine the drug resistance pattern and the role of RhoA in RR. Two regorafenib-resistant cell lines were constructed based on Huh7 and Hep3B cell lines. In vitro and in vivo assays were conducted to study RhoA expression, the activity of Hippo signaling pathway and cancer stem cell (CSC) traits. The data showed that RhoA was highly expressed, Hippo signaling was hypoactivated and CSC traits were more prominent in RR cells. Inhibiting RhoA could reverse RR, and the alliance of RhoA inhibition and regorafenib synergistically attenuated CSC phenotype. Furthermore, inhibiting LARG/RhoA increased Kibra/NF2 complex formation, prevented YAP from shuttling into the nucleus and repressed CD44 mRNA expression. Clinically, the high expression of RhoA correlated with poor prognosis. LARG, RhoA, YAP1 and CD44 show positive correlation with each other. Thus, inhibition of RhoGEF/RhoA has the potential to reverse RR and repress CSC phenotype in HCC.

Simultaneous Activation of Immunogenic Cell Death and cGAS-STING Pathway by Liver- and Mitochondria-Targeted Gold(I) Complexes for Chemoimmunotherapy of Hepatocellular Carcinoma.

Induction of immunogenic cell death (ICD) and activation of the cyclic GMP-AMP synthase stimulator of interferon gene (cGAS-STING) pathway are two potent anticancer immunotherapeutic strategies in hepatocellular carcinoma (HCC). Herein, 12 liver- and mitochondria-targeting gold(I) complexes (9a-9l) were designed and synthesized. The superior complex 9b produced a considerable amount of reactive oxygen species (ROS) and facilitated DNA excretion, the ROS-induced ICD and DNA activated the cGAS-STING pathway, both of which evoked an intense anticancer immune response in vitro and in vivo. Importantly, 9b strongly inhibited tumor growth in a patient-derived xenograft model of HCC. Overall, we present the first case of simultaneous ICD induction and cGAS-STING pathway activation within the same gold-based small molecule, which may provide an innovative strategy for designing chemoimmunotherapies for HCC.

Toxicity study of compound granules of Hedyotis diffusa: Acute toxicity and long-term toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: The clinical efficacy of the hospital preparation compound granules of Hedyotis diffusa (CGHD), which is composed of Hedyotis diffusa Willd, Smilax china L., Solanum lyratum Thunb., has accumulated a good reputation over the past decades. However, because it is a hospital preparation, few researchers have paid attention to it, resulting in a lack of systematic basic research studies. Thus, it is not clear whether there are safety concerns that restrict its clinical application, and toxicological evaluation of CGHD is needed. AIM OF THE STUDY: The aim of this study was to evaluate the safety of CGHD by conducting acute toxicity and long-term toxicity experiments, with the objective of providing evidence for its clinical safety and a theoretical foundation for its clinical application. MATERIALS AND METHODS: KM mice were selected for the acute toxicity experiment and were administered water or CGHD-E 3 times within 24 h. The reactions of the animals to CGHD treatment were observed and recorded within 1 h after administration and then once a day for 14 consecutive days. SD rats were selected to conduct the long-term toxicity experiment. The drug-treated groups were administered different doses of CGHD-E, which were equivalent to 10 times, 20 times and 50 times the clinical dose in humans. The rats were administered the drug for 28 consecutive days. After 28 days, the animals were sacrificed, and routine blood tests, blood coagulation function analysis, liver and kidney function tests, and glycolipid metabolism related tests were conducted. The major organs of the rats were collected to calculate organ coefficients and perform hematoxylin-eosin (HE) staining. RESULTS: In the CGHD-E acute toxicity experiment, the drug-treated groups did not show adverse reactions or poisoning symptoms, and the maximum tolerated dose of CGHD-E in mice was greater than 45.072 g/kg. In the long-term toxicity experiment, drug-treated rats generally exhibited a good condition, but continuous administration decreased on body weight and food intake, especially in male rats. Coagulation function alterations and the impact on the liver during long-term drug administration were also assessed, which should be emphasized in clinical applications. No significant toxic effects were observed according to routine blood tests or test of liver and kidney function, glucose and lipid metabolism, or ion metabolism. CONCLUSIONS: The results of this study showed that CGHD was nontoxic or had low toxicity, providing not only a scientific basis for its clinical application, determining the appropriate clinical dose and monitoring clinical toxicity but also theoretical support for subsequent clinical drug trials.

Performance and mechanism of pilot-scale carbon fibers enhanced ecological floating beds for urban tail water treatment in optimized ecological floating beds water surface coverage.

A pilot-scale carbon fibers enhanced ecological floating beds (CF-EFBs) was constructed. Compared to EFBs without carbon fibers enhancement, CF-EFBs have the better removal of total inorganic nitrogen (TIN), total phosphorus (TP), and chemical oxygen demand (COD), the removal efficiencies were 3.19, 3.49, and 2.74 times higher than EFBs. Throughout the pilot test (under three different coverage rates), the concentrations of COD, TIN and TP of effluent were 18.11 ± 4.52 mgL-1, 1.95 ± 0.92 mgL-1 and 0.13 ± 0.08 mgL-1. Meanwhile, the average removal of TIN, TP and COD from tailwater was 0.96 gm-2d-1, 0.07 gm-2d-1 and 2.37 gm-2d-1 respectively. When the coverage was 30 %, the CF-EFBs had better nitrogen removal effectiveness (TIN purification ability of 1.49 gm-2d-1). The enrichment of denitrifying bacteria, such as Aridibacter, Nitrospira, Povalibacter, and Phaeodactylibacter increased denitrification efficiency. These results verified the feasibility of CF-EFBs in tailwater treatment at pilot-scale, which was of great significance for the practical application of CF-EFBs.

A feedback loop of PPP and PI3K/AKT signal pathway drives regorafenib-resistance in HCC.

BACKGROUND: Hepatocellular carcinoma (HCC) is a principal type of liver cancer with high incidence and mortality rates. Regorafenib is a novel oral multikinase inhibitor for second-line therapy for advanced HCC. However, resistance to regorafenib is gradually becoming a dilemma for HCC and the mechanism remains unclear. In this study, we aimed to reveal the metabolic profiles of regorafenib-resistant cells and the key role and mechanism of the most relevant metabolic pathway in regorafenib resistance. METHODS: Metabolomics was performed to detect the metabolic alteration between drug-sensitive and regorafenib-resistant cells. Colony formation assay, CCK-8 assay and flow cytometry were applied to observe cell colony formation, cell proliferation and apoptosis, respectively. The protein and mRNA levels were detected by western blot and RT-qPCR. Cell lines of Glucose-6-phosphate dehydrogenase(G6PD) knockdown in regorafenib-resistant cells or G6PD overexpression in HCC cell lines were stably established by lentivirus infection technique. G6PD activity, NADPH level, NADPH/NADP+ ratio, the ratio of ROS positive cells, GSH level, and GSH/GSSG ratio were detected to evaluate the anti-oxidative stress ability of cells. Phosphorylation levels of NADK were evaluated by immunoprecipitation. RESULTS: Metabonomics analysis revealed that pentose phosphate pathway (PPP) was the most relevant metabolic pathway in regorafenib resistance in HCC. Compared with drug-sensitive cells, G6PD enzyme activity, NADPH level and NADPH/NADP+ ratio were increased in regorafenib-resistant cells, but the ratio of ROS positive cells and the apoptosis rate under the conditions of oxidative stress were decreased. Furthermore, G6PD suppression using shRNA or an inhibitor, sensitized regorafenib-resistant cells to regorafenib. In contrast, G6PD overexpression blunted the effects of regorafenib to drug-sensitive cells. Mechanistically, G6PD, the rate-limiting enzyme of PPP, regulated the PI3K/AKT activation. Furthermore, PI3K/AKT inhibition decreased G6PD protein expression, G6PD enzymatic activity and the capacity of PPP to anti-oxidative stress possibly by inhibited the expression and phosphorylation of NADK. CONCLUSION: Taken together, a feedback loop of PPP and PI3K/AKT signal pathway drives regorafenib-resistance in HCC and targeting the feedback loop could be a promising approach to overcome drug resistance.

Enhanced electron recovery by optimizing sandwich structure agricultural waste corncob filled anode in microbial electrochemical system to facilitate wastewater denitrification.

Microbial electrochemical system autotrophic denitrification has attracted much attention due to its cost-efficiency and clean advantages. The autotrophic denitrification rate highly depends on the input electrons to the cathode. In this study, agricultural waste corncob was filled into sandwich structure anode as low-cost carbon source for electron production. The COMSOL software was used to guide the construction of sandwich structure anode to control carbon source release and enhance electron collection, including suitable pore size (4 mm) and current collector arrangement (five branches). Optimized sandwich structure anode system with the help of 3D printing obtained a higher denitrification efficiency (21.79 ± 0.22 gNO3--N/m3d) than anodic systems without pore and current collector. Statistical analysis showed that enhanced autotrophic denitrification efficiency was the responsible for enhanced denitrification performance of the optimized anode system. This study provides a strategy to improve the autotrophic denitrification performance of the microbial electrochemical system by optimizing the anode structure.

Pathological characteristics and immune microenvironment of SMARCA4-deficient undifferentiated uterine sarcoma.

SMARCA4-deficient undifferentiated uterine sarcoma (SDUS) is a highly invasive single-gene malignant tumor caused by mutations in the SMARCA4 gene. SDUS has a poor prognosis, with no established treatment strategy at present. Further, there is a lack of relevant research on the role of the immune microenvironment in SDUS worldwide. Here, we report a case of SDUS that was diagnosed and analysed using morphological, immunohistochemical, and molecular detection techniques, along with the analysis of the immune microenvironment. By immunohistochemistry, the tumor cells showed retained INI-1 expression, focal CD10 expression, and loss of BRG1, CK-pan, synaptophysin, desmin, and ER expression. Further, some of the immune cells expressing CD3 and CD8 had infiltrated into the SDUS, but no PD-L1 expression was detected. The multiple immunofluorescent staining results showed that a proportion of the immune cells and SDUS cells expressed CD8/CD68/PD-1/PD-L1. Therefore, our report will help in the diagnostic awareness of SDUS.

Character displacement of egg colors during tinamou speciation.

The divergence of reproductive traits frequently underpins the evolution of reproductive isolation. Here we investigated whether tinamou (Tinamidae) egg colorations function as mating signals that diverged as character displacement (mating signal character displacement hypothesis). We tested three evolutionary predictions behind the hypothesis: (a) egg colors coevolve with known mating signals; (b) signal divergence is associated with divergent habitat adaptation; and (c) sympatric tinamou species with similar songs have different egg colors as character displacement during speciation. We found support for all three predictions. In particular, egg colors coevolved with songs; songs and egg colors coevolved with habitat partitioning; and tinamou species that were likely sympatric with similar songs tended to have different egg colors. In conclusion, the mating signal character displacement hypothesis is well supported in which egg colors serve as mating signals that undergo character displacement during tinamou speciation.

Electrochemical activation of persulfate by Al-doped blue TiO2 nanotubes for the multipath degradation of atrazine.

The combination of electrolysis and persulfate activation (E/PDS) is a cost-effective method for the treatment of refractory organics. However, persulfate is difficult to be activated into radicals at the anode, resulting in insufficient electro-activation efficiency. Herein, Al doped blue TiO2 nanotube electrodes (Al-bTNT) were first employed as cost-effective anode materials to fully activate PDS to radicals. In E/PDS, the kinetic constant of atrazine removal by Al-bTNT (0.048 min-1) substantially outperformed the other anodes, including the blue TiO2 nanotube electrodes (bTNT) (0.024 min-1), Ti4O7 (0.02 min-1), and B doped diamond (BDD) anodes (0.023 min-1). The Al-bTNT-E/PDS exhibited a low energy consumption (EEO = 0.72 kWh m-3) and a high mineralization rate. Based on the results of electron paramagnetic resonance, quenching experiments, and probe experiments, we propose that atrazine degrades in the Al-bTNT-E/PDS system mainly via a novel radical pathway that involves both·OH and SO4·- and the generated SO4·- is responsible for the enhanced removal rate. The oxygen vacancies (VO) generated from interstitial Al may serve as the active sites to adsorb and dissociate the persulfate molecules based on extensive characterizations. The attempt at soil-washing wastewater disposal indicated the synergistic system possessed good potential for future practical application.

Recovery of electron and carbon source from agricultural waste corncob by microbial electrochemical system to enhance wastewater denitrification.

The denitrification process in wastewater treatment plants (WWTPs) is limited by insufficient carbon sources. Agricultural waste corncob was investigated for its feasibility as a low-cost carbon source for efficient denitrification. The results showed that the corncob as the carbon source exhibited a similar denitrification rate (19.01 ± 0.03 gNO3--N/m3d) to that of the traditional carbon source sodium acetate (19.13 ± 0.37 gNO3--N/m3d). When filling corncob into a microbial electrochemical system (MES) three-dimensional anode, the release of corncob carbon sources was well controlled with an improved denitrification rate (20.73 ± 0.20 gNO3--N/m3d). Carbon source and electron recovered from corncob led to autotrophic denitrification and heterotrophic denitrification occurred in the MES cathode, which synergistically improved the denitrification performance of the system. The proposed strategy for enhanced nitrogen removal by autotrophic coupled with heterotrophic denitrification using agricultural waste corncob as the sole carbon source opened up an attractive route for low-cost and safe deep nitrogen removal in WWTPs and resource utilization for agricultural waste corncob.

Construction of a new type of three-dimensional honeycomb-structure anode in microbial electrochemical systems for energy harvesting and pollutant removal.

Electrode materials occupy most of the construction cost of the microbial electrochemical system (MES), and the low mechanical strength and poor electrochemical performance of the commonly used traditional carbon-based materials restrict the promotion and application of this technology. In this study, polymer-based three-dimensional (3D) honeycomb-structure (HS) materials with good mechanical properties were used as supporting materials. Graphene (GR), carbon nanotube (CNT), and polypyrrole (PPy) was separately chosen as a surface conductivity coating layer for preparing MES anodes. The introduction of GR, CNT, and PPy on HS increased surface roughness, hydrophilicity, O and N content, electrochemically active surface area, and decreased charge transfer internal resistance, which promoted the adhesion of microorganisms on their surface and enhanced the extracellular electron transfer process at the electrode/microbe interface. The CNT-HS anode system got the better maximal power density (1700.7 ± 149.0 mW/m2) of the three modified anode systems and 3.60 times that of MES using CC (471.8 ± 27.2 mW/m2) as the anode. The accelerated reactions of the redox species in the outer cell membrane, the promoted electron shuttle secretion, and the enhanced abundance of the tricarboxylic acid cycle-related functional genes in biofilm led to better performance of the CNT-HS anode system. The CNT-HS anode system also exhibited long-term operational stability (>6 months) and a good chemical oxygen demand degradation effect. Furthermore, CNT-HS material exhibited its cost advantage, and its projected cost is estimated to be about $1.8/m2, much lower than the currently used MES anodes ($8.2-548.2/m2). Considering the good mechanical properties, simple preparation process, low manufacturing cost, long-term stability, excellent bio-electrochemical performance, and good pollutant removal ability, HS-based anode has promising potential for high-performance MES in applications.

Dosage compensation in Bombyx mori is achieved by partial repression of both Z chromosomes in males.

SignificanceGenes on sex chromosomes (i.e. human chX) are regulated differently in males and females to balance gene expression levels between sexes (XY vs. XX). This sex-specific regulation is called dosage compensation (DC). DC is achieved by altering the shape and compaction of sex chromosomes specifically in one sex. In this study, we use Oligopaints to examine DC in silkworms. This study visualizes this phenomenon in a species with ZW sex chromosomes, which evolved independently of XY. Our data support a long-standing model for how DC mechanisms evolved across species, and we show potential similarity between DC in silkworms and nematodes, suggesting that this type of DC may have emerged multiple independent times throughout evolution.

Temporal inhibition of chromatin looping and enhancer accessibility during neuronal remodeling.

During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. The chromatin insulator-associated protein Alan Shepard (Shep) promotes Drosophila post-mitotic neuronal remodeling by repressing transcription of master developmental regulators, such as brain tumor (brat), specifically in maturing neurons. Since insulator proteins can promote looping, we hypothesized that Shep antagonizes brat promoter interaction with an as yet unidentified enhancer. Using chromatin conformation capture and reporter assays, we identified two enhancer regions that increase in looping frequency with the brat promoter specifically in pupal brains after Shep depletion. The brat promoters and enhancers function independently of Shep, ruling out direct repression of these elements. Moreover, ATAC-seq in isolated neurons demonstrates that Shep restricts chromatin accessibility of a key brat enhancer as well as other enhancers genome-wide in remodeling pupal but not larval neurons. These enhancers are enriched for chromatin targets of Shep and are located at Shep-inhibited genes, suggesting direct Shep inhibition of enhancer accessibility and gene expression during neuronal remodeling. Our results provide evidence for temporal regulation of chromatin looping and enhancer accessibility during neuronal maturation.

Simultaneous electricity generation and eutrophic water treatment utilizing iron coagulation cell with nitrification and denitrification biocathodes.

Anthropogenic nutrients released into water induce eutrophication and threaten aquatic life and human health. In this study, an Fe anode coagulation cell with nitrification and denitrification biocathodes was constructed for power generation and algae and nutrient removal. The nitrification and denitrification biocathodes achieved maximum power densities of 6.0 and 6.6 W/m3, respectively. The algae (99.2 ± 0.5%), phosphate (97.4 ± 0.6%), and ammonia (23.1 ± 0.2%) were removed by a spontaneous electrocoagulation process in the anode chamber. In the nitrification biocathode chamber, 95.3 ± 1.4% of the ammonia was oxidized within 6 h, and 88.2 ± 2.5% of the nitrate was removed in 10 h in the denitrification biocathode chamber. The microbial community analysis revealed that ammonia removal was attributed to nitrifying bacteria, including Acinetobacter sp., Phycisphaera sp., and Nitrosomonas sp., and the dominant denitrifying bacteria in the denitrifying biocathode chamber were Planococcus sp., Exiguobacterium sp., and Lysinibacillus sp. In this study, the combination of Fe anodes and biocathodes is shown to afford an efficient method for the simultaneous algae and nutrient removal and power generation.

Non-Coding RNA m6A Modification in Cancer: Mechanisms and Therapeutic Targets.

Recently, N6-methyl-adenosine (m6A) ribonucleic acid (RNA) modification, a critical and common internal RNA modification in higher eukaryotes, has generated considerable research interests. Extensive studies have revealed that non-coding RNA m6A modifications (e.g. microRNAs, long non-coding RNAs, and circular RNAs) are associated with tumorigenesis, metastasis, and other tumour characteristics; in addition, they are crucial molecular regulators of cancer progression. In this review, we discuss the relationship between non-coding RNA m6A modification and cancer progression from the perspective of various cancers. In particular, we focus on important mechanisms in tumour progression such as proliferation, apoptosis, invasion and metastasis, tumour angiogenesis. In addition, we introduce clinical applications to illustrate more vividly that non-coding RNA m6A modification has broad research prospects. With this review, we aim to summarize the latest insights and ideas into non-coding RNA m6A modification in cancer progression and targeted therapy, facilitating further research.

Hydrophilic graphene aerogel anodes enhance the performance of microbial electrochemical systems.

The hydrophilic three-dimensional (3D) structure of graphene materials was produced with reducing agent-ethylene glycol through hydrothermal reduction. Numerous microorganisms with diverse community structure were established in anode surface, as the hydrophilicity of the graphene anode increased; more populations of Proteobacteria and Firmicutes families were identified in a higher hydrophilic anode. In addition, the start-up time of a microbial fuel cell (MFC) equipped with hydrophilic 3D graphene anode was only 43 h, which is much shorter than traditional 3D graphene-based anode systems. The most hydrophilic anode exhibited the maximal power density of 583.8 W m-3, 5 times larger than the least hydrophilic one. The content of oxygen in graphene materials improving hydrophilicity would play an important role in enhancing power density. This study proves that hydrophilic 3D graphene materials as the anode can improve MFC performance and start-up time.

Function and regulation of chromatin insulators in dynamic genome organization.

Chromatin insulators are DNA-protein complexes that play a crucial role in regulating chromatin organization. Within the past two years, a plethora of genome-wide conformation capture studies have helped reveal that insulators are necessary for proper genome-wide organization of topologically associating domains, which are formed in a manner distinct from that of compartments. These studies have also provided novel insights into the mechanics of how CTCF/cohesin-dependent loops form in mammals, strongly supporting the loop extrusion model. In combination with single-cell imaging approaches in both Drosophila and mammals, the dynamics of insulator-mediated chromatin interactions are also coming to light. Insulator-dependent structures vary across individual cells and tissues, highlighting the need to study the regulation of insulators in particular temporal and spatial contexts throughout development.