Evaluation of Compound Activity in Primary Human Intestinal Organoids Using Gene Expression and Histology.

Human intestinal organoids have enabled performance of functional epithelial studies and modeling of human diseases of the intestine. This unit describes 1) a method to isolate and culture crypts from human intestinal tissue, 2) use of combinatorial methods to expand stem cell-enriched spheroids and differentiate them into organoids composed of various intestinal epithelial cell types, and 3) methods to stimulate these organoids with and measure their responsiveness to external stimuli. To validate the differentiation, organoids can be stained to qualitatively evaluate the presence of colonic crypt morphology and specialized epithelial cell markers. These organoids are responsive to challenge with tumor necrosis factor α (TNFα), resulting in cytokine-induced apoptosis. TNFα-driven apoptosis can be blocked by a small-molecule inhibitor of Ire1α (4µ8C), an endoplasmic-reticulum stress sensor. This is one example of how the human intestinal organoid model can be a powerful tool to elucidate important biological pathways involved in human disease in intestinal epithelial cells. © 2019 by John Wiley & Sons, Inc.

RORγt and RORα signature genes in human Th17 cells.

RORγt and RORα are transcription factors of the RAR-related orphan nuclear receptor (ROR) family. They are expressed in Th17 cells and have been suggested to play a role in Th17 differentiation. Although RORγt signature genes have been characterized in mouse Th17 cells, detailed information on its transcriptional control in human Th17 cells is limited and even less is known about RORα signature genes which have not been reported in either human or mouse T cells. In this study, global gene expression of human CD4 T cells activated under Th17 skewing conditions was profiled by RNA sequencing. RORγt and RORα signature genes were identified in these Th17 cells treated with specific siRNAs to knock down RORγt or RORα expression. We have generated selective small molecule RORγt modulators and they were also utilized as pharmacological tools in RORγt signature gene identification. Our results showed that RORγt controlled the expression of a very selective number of genes in Th17 cells and most of them were regulated by RORα as well albeit a weaker influence. Key Th17 genes including IL-17A, IL-17F, IL-23R, CCL20 and CCR6 were shown to be regulated by both RORγt and RORα. Our results demonstrated an overlapping role of RORγt and RORα in human Th17 cell differentiation through regulation of a defined common set of Th17 genes. RORγt as a drug target for treatment of Th17 mediated autoimmune diseases such as psoriasis has been demonstrated recently in clinical trials. Our results suggest that RORα could be involved in same disease mechanisms and gene signatures identified in this report could be valuable biomarkers for tracking the pharmacodynamic effects of compounds that modulate RORγt or RORα activities in patients.

Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis.

The IL-23/IL-17 pathway is implicated in autoimmune diseases, particularly psoriasis, where biologics targeting IL-23 and IL-17 have shown significant clinical efficacy. Retinoid-related orphan nuclear receptor gamma t (RORγt) is required for Th17 differentiation and IL-17 production in adaptive and innate immune cells. We identified JNJ-54271074, a potent and highly-selective RORγt inverse agonist, which dose-dependently inhibited RORγt-driven transcription, decreased co-activator binding and promoted interaction with co-repressor protein. This compound selectively blocked Th17 differentiation, significantly reduced IL-17A production from memory T cells, and decreased IL-17A- and IL-22-producing human and murine γδ and NKT cells. In a murine collagen-induced arthritis model, JNJ-54271074 dose-dependently suppressed joint inflammation. Furthermore, JNJ-54271074 suppressed IL-17A production in human PBMC from rheumatoid arthritis patients. RORγt-deficient mice showed decreased IL-23-induced psoriasis-like skin inflammation and cytokine gene expression, consistent with dose-dependent inhibition in wild-type mice through oral dosing of JNJ-54271074. In a translational model of human psoriatic epidermal cells and skin-homing T cells, JNJ-54271074 selectively inhibited streptococcus extract-induced IL-17A and IL-17F. JNJ-54271074 is thus a potent, selective RORγt modulator with therapeutic potential in IL-23/IL-17 mediated autoimmune diseases.

Synthesis of high quality two-dimensional materials via chemical vapor deposition.

Two-dimensional (2D) materials have attracted much attention due to their unique properties and great potential in various applications. Controllable synthesis of 2D materials with high quality and high efficiency is essential for their large scale applications. Chemical vapor deposition (CVD) has been one of the most important and reliable techniques for the synthesis of 2D materials. In this perspective, the recent advances in the CVD growth of three typical types of two-dimensional materials, graphene, boron nitride and transition metal dichalcogenides (TMDs), are briefly introduced. Large area preparation, single crystal growth and some mechanistic insight are discussed with details. Finally we give a brief comment on the challenges of CVD growth of 2D materials.

Two dimensional nanosheets as conductive, flexible elements in biomaterials.

Since the discovery of graphene in 2004, two-dimensional (2D) nanostructures have been attracting tremendous interest for a variety of applications, including bio-inspired applications, due to their fascinating electronic, mechanical and optical properties. Especially, graphene and other 2D or quasi-2D nanostructures show excellent conductivity and flexibility. Herein, we highlight the recent impressive progress concerning the use of two representative types of 2D and quasi-2D nanostructures, graphene-based nanosheets and ultrathin polymeric nanosheets, as conductive or/and flexible elements to engineer three dimensional (3D) tissues. The results feature the unique potential of these 2D and quasi-2D nanostructures in the biomaterial tissue engineering research field.

Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation.

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7ß, 27-dihydroxycholesterol (7ß, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7ß, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7ß, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

3,5-Dihydroxybenzoic acid, a specific agonist for hydroxycarboxylic acid 1, inhibits lipolysis in adipocytes.

Niacin raises high-density lipoprotein and lowers low-density lipoprotein through the activation of the ß-hydroxybutyrate receptor hydroxycarboxylic acid 2 (HCA2) (aka GPR109a) but with an unwanted side effect of cutaneous flushing caused by vascular dilation because of the stimulation of HCA2 receptors in Langerhans cells in skin. HCA1 (aka GPR81), predominantly expressed in adipocytes, was recently identified as a receptor for lactate. Activation of HCA1 in adipocytes by lactate results in the inhibition of lipolysis, suggesting that agonists for HCA1 may be useful for the treatment of dyslipidemia. Lactate is a metabolite of glucose, suggesting that HCA1 may also be involved in the regulation of glucose metabolism. The low potency of lactate to activate HCA1, coupled with its fast turnover rate in vivo, render it an inadequate tool for studying the biological role of lactate/HCA1 in vivo. In this article, we demonstrate the identification of 3-hydroxybenzoic acid (3-HBA) as an agonist for both HCA2 and HCA1, whereas 3,5-dihydroxybenzoic acid (3,5-DHBA) is a specific agonist for only HCA1 (EC(50) ∼150 µM). 3,5-DHBA inhibits lipolysis in wild-type mouse adipocytes but not in HCA1-deficient adipocytes. Therefore, 3,5-DHBA is a useful tool for the in vivo study of HCA1 function and offers a base for further HCA1 agonist design. Because 3-HBA and 3,5-DHBA are polyphenolic acids found in many natural products, such as fruits, berries, and coffee, it is intriguing to speculate that other heretofore undiscovered natural substances may have therapeutic benefits.

Multiwalled carbon nanotubes anchored with SnS2 nanosheets as high-performance anode materials of lithium-ion batteries.

A nanocomposite of multiwalled carbon nanotubes (MWCNTs) anchored with SnS(2) nanosheets (NS) (SnS(2) NS@MWCNTs coaxial nanocables) has been synthesized through a simple solution-based method at room temperature. The synthetic mechanism of these intriguing nanocomposites is proposed as electrostatic attraction between tin ions and MWCNTs, followed by the nucleation and two-dimensional growth of SnS(2). The as-synthesized SnS(2) NS@MWCNTs coaxial nanocables have been applied as anode materials for lithium-ion batteries, which show better lithium storage performance compared to pure SnS(2) nanosheets and MWCNTs. The combination of MWCNTs that can hinder the agglomeration and enhance electronic conductivity of the active materials might be responsible for the enhanced cyclic performance.

Study of GPR81, the lactate receptor, from distant species identifies residues and motifs critical for GPR81 functions.

Receptors from distant species may have conserved functions despite significant differences in protein sequences. Whereas the noncritical residues are often changed in distant species, the amino acids critical in receptor functions are often conserved. Studying the conserved residues between receptors from distant species offers valuable information to probe the roles of residues in receptor function. We identified two zebrafish receptors (zGPR81-1 and zGPR81-2) that show approximately 60% identity to human GPR81, GPR109a, and GPR109b but respond only to l-lactate and not to the GPR109a ligands. Protein sequence comparison among zebrafish GPR81s, mammalian GPR81s, GPR109a, and GPR109b identified a common structure (six Cys residues at the extracellular domains that potentially form three disulfide bonds) in this subfamily of receptors. In addition, a number of residues conserved in all GPR81s but not in GPR109s have been identified. Furthermore, we identified a conserved motif, C165-E166-S167-F168, at the second extracellular loop of GPR81. Using site-directed mutagenesis, we showed that Arg71 at the transmembrane domain 2 is very critical for GPR81 function. In addition, we demonstrated that the C165-E166-S167-F168 motif at the second extracellular loop is critical for GPR81 function, and the conserved six Cys residues at the extracellular regions are necessary for GPR81 function. It is important to mention that for those residues important for GPR81 function, the corresponding residues or motifs in GPR109a are also critical for GPR109a function. These findings help us better understand the interaction between lactate and GPR81 and provide useful information for GPR81 ligand design.

Oxysterols direct B-cell migration through EBI2.

EBI2 (also called GPR183) is an orphan G-protein-coupled receptor that is highly expressed in spleen and upregulated upon Epstein-Barr-virus infection. Recent studies indicated that this receptor controls follicular B-cell migration and T-cell-dependent antibody production. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol metabolism. The biological effects of oxysterols have largely been credited to the activation of nuclear hormone receptors. Here we isolate oxysterols from porcine spleen extracts and show that they are endogenous ligands for EBI2. The most potent ligand and activator is 7α,25-dihydroxycholesterol (OHC), with a dissociation constant of 450 pM for EBI2. In vitro, 7α,25-OHC stimulated the migration of EBI2-expressing mouse B and T cells with half-maximum effective concentration values around 500 pM, but had no effect on EBI2-deficient cells. In vivo, EBI2-deficient B cells or normal B cells desensitized by 7α,25-OHC pre-treatment showed reduced homing to follicular areas of the spleen. Blocking the synthesis of 7α,25-OHC in vivo with clotrimazole, a CYP7B1 inhibitor, reduced the content of 7α,25-OHC in the mouse spleen and promoted the migration of adoptively transferred pre-activated B cells to the T/B boundary (the boundary between the T-zone and B-zone in the spleen follicle), mimicking the phenotype of pre-activated B cells from EBI2-deficient mice. Our results show an unexpected causal link between EBI2, an orphan G-protein-coupled receptor controlling B-cell migration, and the known immunological effects of certain oxysterols, thus uncovering a previously unknown role for this class of molecules.

Assembling CoSn3 nanoparticles on multiwalled carbon nanotubes with enhanced lithium storage properties.

CoSn(3) nanoparticles have been successfully assembled on noncovalently poly(diallyldimethylammonium chloride)-functionalized multiwalled carbon nanotubes (MWCNTs) via a chemical reduction method in a polyol system. The influences of the surface functionality and the reaction temperature on the synthesis of uniform CoSn(3)-MWCNTs nanohybrids have been investigated. The as-synthesized CoSn(3)-MWCNTs nanohybrids have been applied as anodes for lithium-ion batteries, and show better lithium storage performance compared to the bare CoSn(3) nanoparticles and MWCNTs. The combining of MWCNTs that can hinder the agglomeration and enhance the electronic conductivity of the active materials is responsible for the enhanced cyclic performance.

Porous ZnCo2O4 nanowires synthesis via sacrificial templates: high-performance anode materials of Li-ion batteries.

A simple microemulsion-based method has been developed to synthesize ZnCo(2)(C(2)O(4))(3) nanowires that can be transformed to porous ZnCo(2)O(4) nanowires under annealing conditions. The morphology of porous ZnCo(2)O(4) nanowires can be tuned by the initial ZnCo(2)(C(2)O(4))(3) nanowires and the annealing temperatures. The as-synthesized porous ZnCo(2)O(4) nanowires have been applied as anode materials of Li-ion batteries, which show superior capacity and cycling performance. The porous one-dimensional (1D) nanostructures and large surface area are responsible for the superior performance. Moreover, it is indicated that porous ZnCo(2)O(4) nanowires synthesized at low annealing temperature (500 °C) show larger capacity and better cycling performance than that prepared at high annealing temperature (700 °C), because of their higher porosity and larger surface area.

Carbon-coated SnO2 nanotubes: template-engaged synthesis and their application in lithium-ion batteries.

This paper reports the synthesis of carbon-coated SnO2 (SnO2-C) nanotubes through a simple glucose hydrothermal and subsequent carbonization approach by using Sn nanorods as sacrificial templates. The as-synthesized SnO2-C nanotubes have been applied as anode materials for lithium-ion batteries, which exhibit improved cyclic performance compared to pure SnO2 nanotubes. The hollow nanostructure, together with the carbon matrix which has good buffering effect and high electronic conductivity, can be responsible for the improved cyclic performance.

Mutagenesis studies of neuropeptide S identify a suitable peptide tracer for neuropeptide S receptor binding studies and peptides selectively activating the I(107) variant of human neuropeptide S receptor.

Neuropeptide S and its receptor represent a novel neurotransmitter system mainly expressed in the brain. A single nucleotide polymorphism in the first extracellular loop (I107) increases the potency of neuropeptide S and has been identified for both the human neuropeptide S receptor short (A) and long (B) C-terminal forms. Preliminary human genetic studies link this polymorphism to asthma, panic disorders and altered sleep behavior. No polymorphism or splice variants have been reported for the rat neuropeptide S receptor, however it carries an isoleucine at position 107. To identify a suitable tracer for neuropeptide S receptor binding and investigate the role of specific amino acids within neuropeptide S we carried out mutagenesis of the peptide and assessed the ability of the mutations to stimulate calcium release in HEK293 cells expressing human neuropeptide S receptor variants (A, B, AI(107), BI(107)) and rat neuropeptide S receptor. Replacement of threonine at position 8 by arginine and methionine at position 10 by tyrosine resulted in a mutant peptide slightly more potent on all neuropeptide S receptor variants compared to neuropeptide S and more importantly the iodinated mutant peptide was found to be a suitable tracer for binding studies with improved signal to noise ratio and stability compared to [(125)I-Y(10)] neuropeptide S. Replacement of serine at position 1 of neuropeptide S peptide by arginine resulted in a complete loss of potency for the neuropeptide S receptor (long and short form) but not for the I(107) receptor variants (long and short) or rat neuropeptide S receptor.

Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81.

Lactic acid is a well known metabolic by-product of intense exercise, particularly under anaerobic conditions. Lactate is also a key source of energy and an important metabolic substrate, and it has also been hypothesized to be a signaling molecule directing metabolic activity. Here we show that GPR81, an orphan G-protein-coupled receptor highly expressed in fat, is in fact a sensor for lactate. Lactate activates GPR81 in its physiological concentration range of 1-20 mM and suppresses lipolysis in mouse, rat, and human adipocytes as well as in differentiated 3T3-L1 cells. Adipocytes from GPR81-deficient mice lack an antilipolytic response to lactate but are responsive to other antilipolytic agents. Lactate specifically induces internalization of GPR81 after receptor activation. Site-directed mutagenesis of GPR81 coupled with homology modeling demonstrates that classically conserved key residues in the transmembrane binding domains are responsible for interacting with lactate. Our results indicate that lactate suppresses lipolysis in adipose tissue through a direct activation of GPR81. GPR81 may thus be an attractive target for the treatment of dyslipidemia and other metabolic disorders.

Identification of the domains in RXFP4 (GPCR142) responsible for the high affinity binding and agonistic activity of INSL5 at RXFP4 compared to RXFP3 (GPCR135).

Relaxin-3 is a potent agonist for both G-protein coupled receptors (GPCR) RXFP3 (also known as GPCR135) and RXFP4 (also known as GPCR142) while insulin-like peptides 5 (INSL5) is a selective RXFP4 agonist. INSL5 is also a weak (low affinity) RXFP3 antagonist. RXFP3 and RXFP4 share about 50% homology. We have used gain-of-function (RXFP3 --> RXFP4) and loss-of-function (RXFP4 --> RXFP3) chimeras to identify the domains critical for the binding and activation induced by INSL5. Replacing extracellular loop (EL) 1 or EL3 of RXFP3 with the corresponding domains from RXFP4 does not change the RXFP3 pharmacological profile. Exchanging the N-terminus and EL2 of RXFP3 with these of RXFP4 results in a chimeric receptor (CR5) with a high affinity for INSL5. However, in contrast to native RXFP4, INSL5 does not elicit an agonist response from CR5. Conversely, replacing the N-terminus and EL2 of RXFP4 with counterparts from RXFP3 (CR15) results in a chimeric receptor for which relaxin-3 and INSL5 are high and low affinity agonists, respectively. Further mutagenesis studies indicate that transmembrane (TM) domains 2, 3 and 5 of RXFP4 are critical determinants of functional receptor activation by INSL5. Replacement of TM2, 3, and 5 of RXFP3 with equivalent domains from RXFP4 results in a chimeric receptor that can be activated by INSL5. These results suggest that the N-terminus and EL2 domains of RXFP3 and RXFP4 are involved in ligand binding while TM2, 3, and 5 are critical for receptor activation.

Identification and pharmacological characterization of prokineticin 2 beta as a selective ligand for prokineticin receptor 1.

Prokineticins 1 and 2 (PK1 and PK2) have been recently identified from humans and other mammals and play multiple functional roles. PK proteins are ligands for two G protein-coupled receptors, PK receptor 1 (PKR1) and PK receptor 2 (PKR2). Here, we report the molecular cloning and pharmacological characterization of an alternatively spliced product of the PK2 gene encoding 21 additional amino acids compared with PK2, designated PK2L (for PK2 long form). PK2L mRNA is broadly expressed, as is PK2. However, PK2L mRNA expression is lower in brain, undetectable in kidney, and much higher in lung and spleen than that of PK2. We expressed PK2L in mammalian cells and characterized the resulting peptide in comparison with PK1 and PK2. Biochemical characterization indicates that secreted PK2L protein is processed into a smaller peptide by proteolytic cleavage. We designate this smaller form of peptide as PK2beta. Coexpression of furin with PK2L significantly increased the PK2beta processing efficiency. Functional studies showed that PK1, PK2, and PK2beta stimulate intracellular Ca(2+) responses in PKR1-expressing cells with similar potencies. However, the PK2beta stimulus of Ca(2+) responses in PKR2-expressing cells is at least 10-fold less potent than that of PK1 or PK2. Differences in receptor selectivity combined with differential tissue expression patterns suggest PK2 and PK2beta might have different functions in vivo. PKRs have been reported to couple to G(q) and G(i) proteins. In this report, we show that PKs not only stimulate Ca(2+) mobilization but also induce cAMP accumulation in PKR-expressing cells.

Single-cell laser-capture microdissection and RNA amplification.

Generating gene-expression profiles from laser-captured cells requires the successful combination of laser-capture microdissection, RNA extraction, RNA amplification, and microarray analysis. To permit single-cell gene-expression profiling, the RNA amplification method has to be sufficiently powerful to bridge the gap between the amount of RNA available from a single cell to what is required by the microarray, a gap that spans 5 to 6 orders of magnitude. This chapter focuses on the amplification of RNA using a two-round T7 RNA amplification method. The protocols described are adapted for laser-captured material and have been used to generate gene expression profiles from single laser-captured cells.

Single-cell microarray analysis in hippocampus CA1: demonstration and validation of cellular heterogeneity.

Laser capture microdissection in combination with microarrays allows for the expression analysis of thousands of genes in selected cells. Here we describe single-cell gene expression profiling of CA1 neurons in the rat hippocampus using a combination of laser capture, T7 RNA amplification, and cDNA microarray analysis. Subsequent cluster analysis of the microarray data identified two different cell types: pyramidal neurons and an interneuron. Cluster analysis also revealed differences among the pyramidal neurons, indicating that even a single cell type in vivo is not a homogeneous population of cells at the gene expression level. Microarray data were confirmed by quantitative RT-PCR and in situ hybridization. We also report on the reproducibility and sensitivity of this combination of methods. Single-cell gene expression profiling offers a powerful tool to tackle the complexity of the mammalian brain.