Fusobacterium nucleatum infection modulates the transcriptome and epigenome of HCT116 colorectal cancer cells in an oxygen-dependent manner.

Fusobacterium nucleatum, a gram-negative oral bacterium, has been consistently validated as a strong contributor to the progression of several types of cancer, including colorectal (CRC) and pancreatic cancer. While previous in vitro studies have shown that intracellular F. nucleatum enhances malignant phenotypes such as cell migration, the dependence of this regulation on features of the tumor microenvironment (TME) such as oxygen levels are wholly uncharacterized. Here we examine the influence of hypoxia in facilitating F. nucleatum invasion and its effects on host responses focusing on changes in the global epigenome and transcriptome. Using a multiomic approach, we analyze epigenomic alterations of H3K27ac and global transcriptomic alterations sustained within a hypoxia and normoxia conditioned CRC cell line HCT116 at 24 h following initial infection with F. nucleatum. Our findings reveal that intracellular F. nucleatum activates signaling pathways and biological processes in host cells similar to those induced upon hypoxia conditioning in the absence of infection. Furthermore, we show that a hypoxic TME favors F. nucleatum invasion and persistence and therefore infection under hypoxia may amplify malignant transformation by exacerbating the effects induced by hypoxia alone. These results motivate future studies to investigate host-microbe interactions in tumor tissue relevant conditions that more accurately define parameters for targeted cancer therapies.

Antipsychotic-induced epigenomic reorganization in frontal cortex of individuals with schizophrenia.

Genome-wide association studies have revealed >270 loci associated with schizophrenia risk, yet these genetic factors do not seem to be sufficient to fully explain the molecular determinants behind this psychiatric condition. Epigenetic marks such as post-translational histone modifications remain largely plastic during development and adulthood, allowing a dynamic impact of environmental factors, including antipsychotic medications, on access to genes and regulatory elements. However, few studies so far have profiled cell-specific genome-wide histone modifications in postmortem brain samples from schizophrenia subjects, or the effect of antipsychotic treatment on such epigenetic marks. Here, we conducted ChIP-seq analyses focusing on histone marks indicative of active enhancers (H3K27ac) and active promoters (H3K4me3), alongside RNA-seq, using frontal cortex samples from antipsychotic-free (AF) and antipsychotic-treated (AT) individuals with schizophrenia, as well as individually matched controls (n=58). Schizophrenia subjects exhibited thousands of neuronal and non-neuronal epigenetic differences at regions that included several susceptibility genetic loci, such as NRG1, DISC1, and DRD3. By analyzing the AF and AT cohorts separately, we identified schizophrenia-associated alterations in specific transcription factors, their regulatees, and epigenomic and transcriptomic features that were reversed by antipsychotic treatment; as well as those that represented a consequence of antipsychotic medication rather than a hallmark of schizophrenia in postmortem human brain samples. Notably, we also found that the effect of age on epigenomic landscapes was more pronounced in frontal cortex of AT-schizophrenics, as compared to AF-schizophrenics and controls. Together, these data provide important evidence of epigenetic alterations in the frontal cortex of individuals with schizophrenia, and remark for the first time on the impact of age and antipsychotic treatment on chromatin organization.

Epigenomic tomography for probing spatially defined chromatin state in the brain.

Spatially resolved epigenomic profiling is critical for understanding biology in the mammalian brain. Single-cell spatial epigenomic assays were developed recently for this purpose, but they remain costly and labor intensive for examining brain tissues across substantial dimensions and surveying a collection of brain samples. Here, we demonstrate an approach, epigenomic tomography, that maps spatial epigenomes of mouse brain at the scale of centimeters. We individually profiled neuronal and glial fractions of mouse neocortex slices with 0.5 mm thickness. Tri-methylation of histone 3 at lysine 27 (H3K27me3) or acetylation of histone 3 at lysine 27 (H3K27ac) features across these slices were grouped into clusters based on their spatial variation patterns to form epigenomic brain maps. As a proof of principle, our approach reveals striking dynamics in the frontal cortex due to kainic-acid-induced seizure, linked with transmembrane ion transporters, exocytosis of synaptic vesicles, and secretion of neurotransmitters. Epigenomic tomography provides a powerful and cost-effective tool for characterizing brain disorders based on the spatial epigenome.

Genetic regulation of cell-type specific chromatin accessibility shapes the etiology of brain diseases.

Nucleotide variants in cell type-specific gene regulatory elements in the human brain are major risk factors of human disease. We measured chromatin accessibility in sorted neurons and glia from 1,932 samples of human postmortem brain and identified 34,539 open chromatin regions with chromatin accessibility quantitative trait loci (caQTL). Only 10.4% of caQTL are shared between neurons and glia, supporting the cell type specificity of genetic regulation of the brain regulome. Incorporating allele specific chromatin accessibility improves statistical fine-mapping and refines molecular mechanisms underlying disease risk. Using massively parallel reporter assays in induced excitatory neurons, we screened 19,893 brain QTLs, identifying the functional impact of 476 regulatory variants. Combined, this comprehensive resource captures variation in the human brain regulome and provides novel insights into brain disease etiology.

Preparation and Characterization of a Janus Membrane with an "Integrated" Structure and Adjustable Hydrophilic Layer Thickness.

Oil-water emulsions are types of wastewater that are difficult to treat. A polyvinylidene fluoride hydrophobic matrix membrane was modified using a hydrophilic polymer, poly(vinylpyrrolidone-vinyltriethoxysilane), to form a representative Janus membrane with asymmetric wettability. The performance parameters of the modified membrane, such as the morphological structure, the chemical composition, the wettability, the hydrophilic layer thickness, and the porosity, were characterized. The results showed that the hydrolysis, migration, and thermal crosslinking of the hydrophilic polymer in the hydrophobic matrix membrane contributed to an effective hydrophilic layer on the surface. Thus, a Janus membrane with unchanged membrane porosity, a hydrophilic layer with controllable thickness, and hydrophilic/hydrophobic layer "structural integration" was successfully prepared. The Janus membrane was used for the switchable separation of oil-water emulsions. The separation flux of the oil-in-water emulsions on the hydrophilic surface was 22.88 L·m-2·h-1 with a separation efficiency of up to 93.35%. The hydrophobic surface exhibited a separation flux of 17.45 L·m-2·h-1 with a separation efficiency of 91.47% for the water-in-oil emulsions. Compared to the lower flux and separation efficiency of purely hydrophobic and hydrophilic membranes, the Janus membrane exhibited better separation and purification effects for both oil-water emulsions.

Three-dimensional genome rewiring in loci with human accelerated regions.

Human accelerated regions (HARs) are conserved genomic loci that evolved at an accelerated rate in the human lineage and may underlie human-specific traits. We generated HARs and chimpanzee accelerated regions with an automated pipeline and an alignment of 241 mammalian genomes. Combining deep learning with chromatin capture experiments in human and chimpanzee neural progenitor cells, we discovered a significant enrichment of HARs in topologically associating domains containing human-specific genomic variants that change three-dimensional (3D) genome organization. Differential gene expression between humans and chimpanzees at these loci suggests rewiring of regulatory interactions between HARs and neurodevelopmental genes. Thus, comparative genomics together with models of 3D genome folding revealed enhancer hijacking as an explanation for the rapid evolution of HARs.

Transcription factor binding site orientation and order are major drivers of gene regulatory activity.

The gene regulatory code and grammar remain largely unknown, precluding our ability to link phenotype to genotype in regulatory sequences. Here, using a massively parallel reporter assay (MPRA) of 209,440 sequences, we examine all possible pair and triplet combinations, permutations and orientations of eighteen liver-associated transcription factor binding sites (TFBS). We find that TFBS orientation and order have a major effect on gene regulatory activity. Corroborating these results with genomic analyses, we find clear human promoter TFBS orientation biases and similar TFBS orientation and order transcriptional effects in an MPRA that tested 164,307 liver candidate regulatory elements. Additionally, by adding TFBS orientation to a model that predicts expression from sequence we improve performance by 7.7%. Collectively, our results show that TFBS orientation and order have a significant effect on gene regulatory activity and need to be considered when analyzing the functional effect of variants on the activity of these sequences.

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex.

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

Lipopeptide liposomes-loaded hydrogel for multistage transdermal chemotherapy of melanoma.

Transdermal administration of chemotherapeutics into tumor tissues may be an effective treatment to reduce toxic side effects and improve patient compliance for melanoma. Herein, we report a multistage transdermal drug delivery system for chemotherapy of melanoma. In this system, dendritic lipopeptide (DLP) modified multistage targeted liposomes (Mtlip) were incorporated into the hydrogel matrix to achieve localized and sustained drug release; Ultra-deformability of Mtlip can pass through dense stratum corneum to the epidermis where melanoma is located; Virus-mimicking Mtlip enhances the payload in tumor tissues by high permeability; The positive charged Mtlip can improve cell uptake efficiency and selectively accumulate into mitochondria to increases toxic. The efficacy of this type of multistage targeted liposomes loaded hydrogel in treating melanoma was systematically evaluated both in vitro and in vivo.

nMOWChIP-seq: low-input genome-wide mapping of non-histone targets.

Genome-wide profiling of interactions between genome and various functional proteins is critical for understanding regulatory processes involved in development and diseases. Conventional assays require a large number of cells and high-quality data on tissue samples are scarce. Here we optimized a low-input chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology for profiling RNA polymerase II (Pol II), transcription factor (TF), and enzyme binding at the genome scale. The new approach produces high-quality binding profiles using 1,000-50,000 cells. We used the approach to examine the binding of Pol II and two TFs (EGR1 and MEF2C) in cerebellum and prefrontal cortex of mouse brain and found that their binding profiles are highly reflective of the functional differences between the two brain regions. Our analysis reveals the potential for linking genome-wide TF or Pol II profiles with neuroanatomical origins of brain cells.

Massively parallel reporter perturbation assays uncover temporal regulatory architecture during neural differentiation.

Gene regulatory elements play a key role in orchestrating gene expression during cellular differentiation, but what determines their function over time remains largely unknown. Here, we perform perturbation-based massively parallel reporter assays at seven early time points of neural differentiation to systematically characterize how regulatory elements and motifs within them guide cellular differentiation. By perturbing over 2,000 putative DNA binding motifs in active regulatory regions, we delineate four categories of functional elements, and observe that activity direction is mostly determined by the sequence itself, while the magnitude of effect depends on the cellular environment. We also find that fine-tuning transcription rates is often achieved by a combined activity of adjacent activating and repressing elements. Our work provides a blueprint for the sequence components needed to induce different transcriptional patterns in general and specifically during neural differentiation.

In-situ construction of heterostructure (Ni, Co)Se2 nanoarrays derived from cone-like ZIF-L for high-performance hybrid supercapacitors.

The construction of heterostructure could enhance the electron transfer efficiency and increase the number of active sites, which can further develop high-performance electrode materials of supercapacitors. Herein, (Ni, Co)Se2 nanorod arrays were prepared based on the NiCo-LDH derived from a conical ZIF-L. Significantly, the single nanorod is composed of interconnected NiSe2 and CoSe2 nanoparticles, the heterostructure can expose higher conductivity, more sufficient redox reaction active sites and larger specific surface area. The as-obtained CF@(Ni, Co)Se2 achieved a high specific capacity of 188.8 mAh g-1 at the current density of 1.0 A g-1 and an outstanding cycling stability with a high capacity retention of 90% after 8000 cycles. Finally, an hybrid supercapacitor device composed of activated carbon (AC) as negative electrode and CF@(Ni, Co)Se2 as positive electrode was designed, which revealed an ideal voltage window of 0-1.6 V and exhibited a great energy density of 36.02 Wh kg-1 at the power density of 800 W kg-1, such surpassing energy storage characteristics evidently testify that (Ni, Co)Se2 nanorod arrays can be as the potential electrode material to promote the development of high-performance supercapacitors.

Multiplexed and Ultralow-Input ChIP-seq Enabled by Tagmentation-Based Indexing and Facile Microfluidics.

Epigenome constitutes an important layer that regulates gene expression and dynamics during development and diseases. Extensive efforts have been made to develop epigenome profiling methods using a low number of cells and with high throughput. Chromatin immunoprecipitation (ChIP) is the most important approach for profiling genome-wide epigenetic changes such as histone modifications. In this report, we demonstrate microfluidic ChIPmentation (mu-CM), a microfluidic technology that enables profiling cell samples that individually do not generate enough ChIP DNA for sequencing library preparation. We used a simple microfluidic device to allow eight samples to be processed simultaneously. The samples were indexed differently using a tagmentation-based approach (ChIPmentation) and then merged for library preparation. A histone modification profile for each individual sample was obtained by demultiplexing the sequencing reads based on the indexes. Our technology allowed profiling 20 cells and is well suited for cell-type-specific studies using low-abundance tissues.

Effects of culture condition on epigenomic profiles of brain tumor cells.

Personalized cancer medicine offers the promise of more effective treatments that are tailored to an individual's own dynamic cancer phenotype. Meanwhile, tissue-engineering approaches to modeling tumors may complement these advances by providing a powerful new approach to understanding the adaptation dynamics occurring during treatment. However, in both of these areas new tools will be required to gain a full picture of the genetic and epigenetic regulators of phenotype dynamics occurring in the small populations of cells that drive resistance. In this study, we perform epigenomic analysis of brain tumor cells that are collected from micro-engineered three-dimensional tumor models, overcoming the challenges associated with the small numbers of cells contained within these micro-tissue niches, in this case collecting ~1,000 cells per sample. Specifically, we use a high-resolution epigenomic analysis method known as microfluidic-oscillatory-washing-based chromatin immunoprecipitation with sequencing (MOWChIP-seq) to analyze histone methylation patterns (H3K4me3). We identified gene loci that are associated with the H3K4me3 modification, which is generally a mark of active transcription. We compared methylation patterns in standard 2D cultures and 3D cultures based on type I collagen hydrogels, under both normoxic and hypoxic conditions. We found that culture dimensionality drastically impacted the H3k4me3 profile and resulted in differential modifications in response to hypoxic stress. Differentially H3K4me3-marked regions under the culture conditions used in this study have important implications for gene expression differences that have been previously observed. In total, our work illustrates a direct connection between cell culture or tissue niche condition and genome-wide alterations in histone modifications, providing the first steps towards analyzing the spatiotemporal variations in epigenetic regulation of cancer cell phenotypes. This study, to our knowledge, also represents the first time broad-spectrum epigenomic analysis has been applied to small cell samples collected from engineered micro-tissues.

Microfluidic epigenomic mapping technologies for precision medicine.

Epigenomic mapping of tissue samples generates critical insights into genome-wide regulations of gene activities and expressions during normal development and disease processes. Epigenomic profiling using a low number of cells produced by patient and mouse samples presents new challenges to biotechnologists. In this review, we first discuss the rationale and premise behind profiling epigenomes for precision medicine. We then examine the existing literature on applying microfluidics to facilitate low-input and high-throughput epigenomic profiling, with emphasis on technologies enabling interfacing with next-generation sequencing. We detail assays on studies of histone modifications, DNA methylation, 3D chromatin structures and non-coding RNAs. Finally, we discuss what the future may hold in terms of method development and translational potential.

CpG oligodeoxynucleotide induces bone marrow precursor cells into myeloid-derived suppressor cells.

Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) perform a number of functions in different immunological settings. In standard in vitro experiments, DCs are produced from mouse bone marrow (BM) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. Our previous study demonstrated that BM precursor cells could differentiate into MDSCs when co-cultured with poly (I:C). In the present study, BM precursor cells cultured in GM-CSF and IL-4 were treated with CpG oligodeoxynucleotide (CpG ODN). We observed that Gr1+CD11b+ cells exhibiting MDSC functions accumulated in the co-culture system. A similar phenomenon was also observed in Listeria monocytogenes-infected mice. In conclusion, we demonstrated that prolonged CpG ODN stimulation could inhibit the development of DCs and induce the differentiation of BM precursor cells into MDSCs.