Single-cell meta-analysis of inflammatory bowel disease with scIBD.

Understanding the heterogeneous intestinal microenvironment is critical to uncover the pathogenesis of inflammatory bowel disease (IBD). Recent advances in single-cell RNA sequencing (scRNA-seq) have identified certain cell types and genes that could contribute to IBD; however, a comprehensively integrated analysis of these scRNA-seq datasets is not yet available. Here we introduce scIBD, a platform for single-cell meta-analysis of IBD with interactive and visualization features, which combines highly curated single-cell datasets in a uniform workflow, enabling identifying rare or less-characterized cell types in IBD and dissecting the commonalities, as well as the differences between ulcerative colitis and Crohn's disease. scIBD also incorporates multifunctional information-including regulon activity, GWAS-implicated risk genes and genes targeted by therapeutics-to infer clinically relevant cell-type specificity. Collectively, scIBD is a user-friendly web-based platform for the community to analyze the transcriptome features and gene regulatory networks associated with the pathogenesis and treatment of IBD at single-cell resolution.

Inhibition of myeloid PD-L1 suppresses osteoclastogenesis and cancer bone metastasis.

Programmed death-ligand 1 (PD-L1) is predominantly expressed in the antigen-presenting cells (APCs) that are originated and are abundant in the bone marrow. The roles of PD-L1 in bone cell differentiation and cancer bone metastasis remain unclear. Here we show that PD-L1 antibody or PD-L1 conditional knockout in the hematopoietic or myeloid lineage suppresses osteoclast differentiation in vitro and in vivo. Bone metastases of breast cancer and melanoma are diminished by PD-L1 antibody or PD-L1 deletion in the myeloid lineage. Transcriptional profiling of bone marrow cells reveals that PD-L1 deletion in the myeloid cells upregulates immune-stimulatory genes, leading to increased macrophage M1 polarization, decreased M2 polarization, enhanced IFNγ signaling, and elevated T cell recruitment and activation. All these alterations result in heightened anti-tumor immunity in the cancer microenvironment. Our findings support PD-L1 antibody as a potent therapy for bone metastasis of breast cancer and melanoma by simultaneously suppressing osteoclast and enhancing immunity.

Fam20C Regulates Bone Resorption and Breast Cancer Bone Metastasis through Osteopontin and BMP4.

Fam20C is a kinase that generates the majority of secreted phosphoproteins and regulates biomineralization. However, its potential roles in bone resorption and breast cancer bone metastasis are unknown. Here we show that Fam20C in the myeloid lineage suppresses osteoclastogenesis and bone resorption, during which, osteopontin (OPN) is the most abundant phosphoprotein secreted in a Fam20C-dependent manner. OPN phosphorylation by Fam20C decreased OPN secretion, and OPN neutralization reduced Fam20C deficiency-induced osteoclast differentiation and bone metastasis. In contrast, Fam20C in breast cancer cells promoted bone metastasis by facilitating the phosphorylation and secretion of BMP4, which in turn enhanced osteoclastogenesis. Mutation of the BMP4 phosphorylation site elevated BMP4 lysosomal degradation and reduced BMP4 secretion. In breast cancer cells, BMP4 depletion or treatment with a BMP4 signaling inhibitor diminished osteoclast differentiation and bone metastasis and abolished Fam20C-mediated regulation of these processes. Collectively, this study discovers distinct roles for Fam20C in myeloid cells and breast cancer cells and highlights OPN and BMP4 as potential therapeutic targets for breast cancer bone metastasis. SIGNIFICANCE: Osteoclastogenesis and bone metastasis are suppressed by myeloid-derived Fam20C, but enhanced by breast cancer-associated Fam20C, uncovering novel Fam20C functions and new therapeutic strategies via targeting Fam20C substrates OPN and BMP4.

Tenovin-1 inhibited dengue virus replication through SIRT2.

Dengue fever is a common arbovirus disease, which has been spread to the entire tropical world. At present, effective drugs for the treatment of dengue fever have not yet appeared, and the dengue vaccines studied in various countries have also experienced severe adverse reactions. Thus it is urgent to find new chemicals against dengue virus. Now we found Sirtuins (SIRTs) were increased during dengue virus infection and tenovin-1, a SIRT1/2 inhibitor, showed an impressive antiviral ability in vitro. In BHK-21 cells, tenovin-1 inhibited the replication of DENV2 with an EC50 at 3.41 ± 1.10 µM, also inhibited other three types of dengue viruses with EC50 at 0.97 ± 1.11 µM, 1.81 ± 1.08 µM, 3.81 ± 1.34 µM respectively. Moreover, the cytopathic effect-induced DENV2 was largely improved by tenovin-1 treatment and the release of progeny viruses was inhibited by tenovin-1 treatment. At the same time, the viral protein level and mRNA level were decreased with tenovin-1 treatment after dengue virus infection. From the drug-addition assay, the tenovin-1 played its antiviral after viral infection, which indicated tenovin-1 was not a microbicide. Apart from its antiviral effect, tenovin-1 inhibited the inflammatory response caused by DENV2, reducing the release of inflammatory factors during viral infection. The antiviral effect of tenovin-1 was abrogated with SIRT agonist or SIRT2 knockdown treatment, which indicated the effect of tenovin-1 was on-target. In conclusion, tenovin-1 was proved to be a promising compound against flavivirus infection through SIRT2, which should be pay more attention for further study.

Brivanib alaninate inhibited dengue virus proliferation through VEGFR2/AMPK pathway.

Dengue virus (DENV) is the most prevalent arthropod-borne viral disease of humans and has a major impact on global public health. There is no clinically approved drugs for DENV infection. Since intracellular VEGFR2 is increased in DENV infected patients, we thus hypothesized that VEGFR2 participated DENV proliferation and its inhibitors could be served as antivirals against DENV. Actually our results showed that VEGFR2 was induced by DENV infection. Also the agonist of VEGFR2, VEGF-A, promoted DENV proliferation. Therefore, we screened the inhibitors of VEGFR2 and found that brivanib alaninate (brivanib) showed the best anti-DENV ability with the lowest cellular cytotoxicity. Mechanically, our results indicated VEGFR2 directly interacted with PTP1B to dephosphorylate AMPK to provide lipid environment for viral replication. However, this effect could be inhibited by brivanib, which significantly reversed the reduction of AMPK phosphorylation caused by DENV infection, thus improving the cellular lipid environment. Moreover, the antiviral effect of brivanib could be reversed by AMPK inhibitor, Compound C. In addition, oral administration of brivianib (20-50 mg/kg/day) clearly improved the survival rate of DENV2 infection, and this effect was abolished in accompanied with Compound C (10mg/kg/day). Collectively, our study disclosed the mechanism of VEGFR2 in DENV2 and evaluated the antiviral ability of brivanib, which deserved more attention for clinical usage in DENV infection.

Drug Repurposing of Itraconazole and Estradiol Benzoate against COVID-19 by Blocking SARS-CoV-2 Spike Protein-Mediated Membrane Fusion.

SARS-CoV-2 caused the emerging epidemic of coronavirus disease in 2019 (COVID-19). To date, there are more than 82.9 million confirmed cases worldwide, there is no clinically effective drug against SARS-CoV-2 infection. The conserved properties of the membrane fusion domain of the spike (S) protein across SARS-CoV-2 make it a promising target to develop pan-CoV therapeutics. Herein, two clinically approved drugs, Itraconazole (ITZ) and Estradiol benzoate (EB), are found to inhibit viral entry by targeting the six-helix (6-HB) fusion core of SARS-CoV-2 S protein. Further studies shed light on the mechanism that ITZ and EB can interact with the heptad repeat 1 (HR1) region of the spike protein, to present anti-SARS-CoV-2 infections in vitro, indicating they are novel potential therapeutic remedies for COVID-19 treatment. Furthermore, ITZ shows broad-spectrum activity targeting 6-HB in the S2 subunit of SARS-CoV and MERS-CoV S protein, inspiring that ITZ have the potential for development as a pan-coronavirus fusion inhibitor.

Natural Product Luteolin Rescues THAP-Induced Pancreatic ß-Cell Dysfunction through HNF4α Pathway.

Endoplasmic reticulum stress (ER stress) plays a main role in pancreatic [Formula: see text]-cell dysfunction and death because of intracellular Ca[Formula: see text] turbulence and inflammation activation. Although several drugs are targeting pancreatic [Formula: see text]-cell to improve [Formula: see text]-cell function, there still lacks agents to alleviate [Formula: see text]-cell ER stress conditions. Therefore we used thapsigargin (THAP) or high glucose (HG) to induce ER stress in [Formula: see text]-cell and aimed to screen natural molecules against ER stress-induced [Formula: see text]-cell dysfunction. Through screening the Traditional Chinese drug library ([Formula: see text] molecules), luteolin was finally discovered to improve [Formula: see text]-cell function. Cellular viability results indicated luteolin reduced the THAP or HG-induced [Formula: see text]-cell death and apoptosis through MTT and flow cytometry assay. Moreover, luteolin improved [Formula: see text]-cell insulin secretion ability under ER stress conditions. Also ER stress-induced intracellular Ca[Formula: see text] turbulence and inflammation activation were inhibited by luteolin treatment. Mechanically, luteolin inhibited HNF4[Formula: see text] signaling, which was induced by ER stress. Moreover, luteolin reduced the transcriptional level of HNF4[Formula: see text] downstream gene, such as Asnk4b and HNF1[Formula: see text]. Conversely HNF4[Formula: see text] knockdown abolished the effect of luteolin on [Formula: see text]-cell using siRNA. These results suggested the protective effect of luteolin on [Formula: see text]-cell was through HNF4[Formula: see text]/Asnk4b pathway. In conclusion, our study discovered that luteolin improved [Formula: see text]-cell function and disclosed the underlying mechanism of luteolin on [Formula: see text]-cell, suggesting luteolin is a promising agent against pancreatic dysfunction.

Retraction Note: miR-34a blocks osteoporosis and bone metastasis by inhibiting osteoclastogenesis and Tgif2.

A Retraction to this paper has been published and can be accessed via a link at the top of the paper.

A series of octahydroquinazoline-5-ones as novel inhibitors against dengue virus.

A series of octahydroquinazoline-5-ones (OHQs 1-50) were designed and synthesized via an improved five-component reaction (5CR). Their bioactivities against dengue virus (DENV) were evaluated by determining lacate dehydrogenase (LDH) in the BHK-21 cells infected with DENV-2. Primary structure-activity relationship showed that six of OHQs with suitable substituents displayed good activities with EC50 = 1.31-1.85 µM. The primary bioactivity mechanism was investigated using the most potent OHQ 23. Experimental results indicate that 23 could efficiently reverse the DENV-2-induced cytopathic effect and suppress the expression of viral structure E protein, but showed no interaction with the MTase and RdRp domain of NS5, a protein plays an important role in viral genome transcription and viral protein translation. The efficient synthetic method, novel structures as DENV inhibitors and good activities are expected to be developed potential DENV inhibitors.

Opposing Functions of BRD4 Isoforms in Breast Cancer.

Bromodomain-containing protein 4 (BRD4) is a cancer therapeutic target in ongoing clinical trials disrupting primarily BRD4-regulated transcription programs. The role of BRD4 in cancer has been attributed mainly to the abundant long isoform (BRD4-L). Here we show, by isoform-specific knockdown and endogenous protein detection, along with transgene expression, the less abundant BRD4 short isoform (BRD4-S) is oncogenic while BRD4-L is tumor-suppressive in breast cancer cell proliferation and migration, as well as mammary tumor formation and metastasis. Through integrated RNA-seq, genome-wide ChIP-seq, and CUT&RUN association profiling, we identify the Engrailed-1 (EN1) homeobox transcription factor as a key BRD4-S coregulator, particularly in triple-negative breast cancer. BRD4-S and EN1 comodulate the extracellular matrix (ECM)-associated matrisome network, including type II cystatin gene cluster, mucin 5, and cathepsin loci, via enhancer regulation of cancer-associated genes and pathways. Our work highlights the importance of targeted therapies for the oncogenic, but not tumor-suppressive, activity of BRD4.

Differential regulation of breast cancer bone metastasis by PARP1 and PARP2.

PARP1 and PARP2 dual inhibitors, such as olaparib, have been recently FDA approved for the treatment of advanced breast and ovarian cancers. However, their effects on bone mass and bone metastasis are unknown. Here we show that olaparib increases breast cancer bone metastasis through PARP2, but not PARP1, specifically in the myeloid lineage, but not in the cancer cells. Olaparib treatment or PARP1/2 deletion promotes osteoclast differentiation and bone loss. Intriguingly, myeloid deletion of PARP2, but not PARP1, increases the population of immature myeloid cells in bone marrow, and impairs the expression of chemokines such as CCL3 through enhancing the transcriptional repression by ß-catenin. Compromised CCL3 production in turn creates an immune-suppressive milieu by altering T cell subpopulations. Our findings warrant careful examination of current PARP inhibitors on bone metastasis and bone loss, and suggest cotreatment with CCL3, ß-catenin inhibitors, anti-RANKL or bisphosphonates as potential combination therapy for PARP inhibitors.

[1,2,4]Triazolo[1,5-a]pyrimidine derivative (Mol-5) is a new NS5-RdRp inhibitor of DENV2 proliferation and DENV2-induced inflammation.

Dengue fever is an acute infectious disease caused by dengue virus (DENV) and transmitted by Aedes mosquitoes. There is no effective vaccine or antiviral drug available to date to prevent or treat dengue disease. Recently, RNA-dependent RNA polymerase (RdRp), a class of polymerases involved in the synthesis of complementary RNA strands using single-stranded RNA, has been proposed as a promising drug target. Hence, we screened new molecules against DENV RdRp using our previously constructed virtual screening method. Mol-5, [1,2,4]triazolo[1,5-a]pyrimidine derivative, was screened out from an antiviral compound library (~8000 molecules). Using biophysical methods, we confirmed the direct interactions between mol-5 and purified DENV RdRp protein. In luciferase assay, mol-5 inhibited NS5-RdRp activity with an IC50 value of 1.28 ± 0.2 µM. In the cell-based cytopathic effect (CPE) assay, mol-5 inhibited DENV2 infectivity with an EC50 value of 4.5 ± 0.08 µM. Mol-5 also potently inhibited DENV2 RNA replication as observed in immunofluorescence assay and qRT-PCR. Both the viral structural (E) and non-structural (NS1) proteins of DENV2 were dose-dependently decreased by treatment with mol-5 (2.5-10 µM). Mol-5 treatment suppressed DENV2-induced inflammation in host cells, but had no direct effect on host defense (JAK/STAT-signaling pathway). These results demonstrate that mol-5 could be a novel RdRp inhibitor amenable for further research and development.

Metabolic Reprogramming in Mitochondria of Myeloid Cells.

The myeloid lineage consists of multiple immune cell types, such as macrophages, monocytes, and dendritic cells. It actively participates in both innate and adaptive immunity. In response to pro- or anti-inflammatory signals, these cells undergo distinct programmed metabolic changes especially in mitochondria. Pro-inflammatory signals induce not only a simple shift from oxidative phosphorylation to glycolysis, but also complicated metabolic alterations during the early and tolerant stages in myeloid cells. In mitochondria, a broken Krebs cycle leads to the accumulation of two metabolites, citrate and succinate, both of which trigger pro-inflammatory responses of myeloid cells. A deficient electron transport chain induces pro-inflammatory responses in the resting myeloid cells while it suppresses these responses in the polarized cells during inflammation. The metabolic reprogramming in mitochondria is also associated with altered mitochondrial morphology. On the other hand, intact oxidative phosphorylation is required for the anti-inflammatory functions of myeloid cells. Fatty acid synthesis is essential for the pro-inflammatory effect and glutamine metabolism in mitochondria exhibits the anti-inflammatory effect. A few aspects of metabolic reprogramming remain uncertain, for example, glycolysis and fatty acid oxidation in anti-inflammation. Overall, metabolic reprogramming is an important element of immune responses in myeloid cells.

GLP-1 Receptor Activation Abrogates ß-Cell Dysfunction by PKA Cα-Mediated Degradation of Thioredoxin Interacting Protein.

Glucagon-like peptide 1 receptor (GLP-1R) agonist (Exendin-4) is a well-known agent used to improve ß-cell dysfunctions via protein kinase A (PKA), but the detailed downstream molecular mechanisms are still elusive. We have now found that PKA Cα mediated- TXNIP phosphorylation and degradation played a vital role in the ß-cell protective role of exendin-4. After PKA activator (Exendin-4 or FSK) treatment, PKA Cα could directly interact with TXNIP by bimolecular fluorescence complementation and Co-IP assays in INS-1 cells. And PKA Cα overexpression decreased TXNIP level, whereas TXNIP level was largely increased in PKA Cα-KO ß-cells by CRISPR-Cas9. Interestingly, TXNIP overexpression or PKA Cα-KO has impaired ß-cell functions, including loss of insulin secretion and activation of inflammation. PKA Cα directly phosphorylated TXNIP at Ser307 and Ser308 positions, leading to its degradation via activation of cellular proteasome pathway. Consistent with this observation, TXNIP (S307/308A) mutant resisted the degradation effects of PKA Cα. However, exendin-4 neither affected TXNIP level in TXNIP (S307/308A) mutant overexpressed ß-cells nor in PKA Cα-KO ß-cells. Moreover, exendin-4 treatment reduced the inflammation gene expression in TXNIP overexpressed ß-cells, but exendin-4 treatment has no effect on the inflammation gene expression in TXNIP (S307/308A) overexpressed ß-cells. In conclusion, our study reveals the integral role of PKA Cα/TXNIP signaling in pancreatic ß-cells and suggests that PKA Cα-mediated TXNIP degradation is vital in ß-cell protective effects of exendin-4.

Molecular determinants for the polarization of macrophage and osteoclast.

Emerging evidence suggest that macrophage and osteoclast are two competing differentiation outcomes from myeloid progenitors. In this review, we summarize recent advances in the understanding of the molecular mechanisms controlling the polarization of macrophage and osteoclast. These include nuclear receptors/transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and estrogen-related receptor α (ERRα), their transcription cofactor PPARγ coactivator 1-ß (PGC-1ß), metabolic factors such as mitochondrial complex I (CI) component NADH:ubiquinone oxidoreductase iron-sulfur protein 4 (Ndufs4), as well as transmembrane receptors such as very-low-density-lipoprotein receptor (VLDLR). These molecular rheostats promote osteoclast differentiation but suppress proinflammatory macrophage activation and inflammation, by acting lineage-intrinsically, systemically or cross generation. These findings provide new insights to the understanding of the interactions between innate immunity and bone remodeling, advancing the field of osteoimmunology.

Author Correction: miR-34a blocks osteoporosis and bone metastasis by inhibiting osteoclastogenesis and Tgif2.

Change history: In this Letter, the citation to 'Fig. 4e, f' in the main text should be 'Fig. 3e, f'. This has not been corrected online.

NR Supplementation During Lactation: Benefiting Mother and Child.

Abnormal nicotinamide adenine dinucleotide (NAD+) metabolism causes a wide spectrum of diseases. A recent study (Cell Rep. 2019;26:969-983) shows that postpartum NAD+ homeostasis is depressed. By restoring NAD+ homeostasis, maternal nicotinamide riboside (NR) supplementation during lactation enhances postpartum weight loss, as well as juvenile development and adult neurogenesis in the offspring.

Q63, a novel DENV2 RdRp non-nucleoside inhibitor, inhibited DENV2 replication and infection.

Dengue virus (DENV) annually infects 400 million people worldwide. Unfortunately, there is lack of widely protective vaccine or drugs against DENV. The viral RNA-dependent RNA polymerase (RdRp) of NS5 protein is highly conserved among different DENV subtypes, thus presenting itself as an attractive target for drug design. In the current research, SPRi was performed to screen compounds against DENV2 RdRp and 5(1H)-Quinazolinone,2-(4-bromophenyl)-2,3,4,6,7,8-hexahydro-7,7-dimethyl-1,3-diphenyl (Q63) was successfully screened out with a KD of 0.9 µM. Then, ITC and molecular docking assay was performed to access the binding mechanism between Q63 and DENV2 RdRp. Meanwhile, Q63 also decreased the intermediate dsRNA production, which was the product of RdRp. Further the antiviral effects of Q63 were evaluated on mosquito C6/36 cells and mammalian BHK-21 cells. Q63 reduced CPE and cell toxicity effect after DENV2 infection on C6/36 and BHK-21 cells, with an EC50 of 2.08 µM. Time of addition assay revealed that Q63 affected the early genome RNA replication stage, including genome RNA replication. In addition, Q63 down-regulated STAT1 phosphorylation, ISG15 and ISG54 after DENV2 infection. In summary, Q63 was found to be a novel RdRp non-nucleoside inhibitor and a potential lead compound for coping with DENV infectious disease in the future.

mTORC1 impedes osteoclast differentiation via calcineurin and NFATc1.

Rapamycins are immunosuppressant and anti-cancer drugs that inhibit the kinase mTOR. Clinically, they often cause bone pain, bone necrosis, and high bone turnover, yet the mechanisms are unclear. Here we show that mTORC1 activity is high in osteoclast precursors but downregulated upon RANKL treatment. Loss-of-function genetic models reveal that while early Raptor deletion in hematopoietic stem cells blunts osteoclastogenesis due to compromised proliferation/survival, late Raptor deletion in osteoclast precursors instead augments osteoclastogenesis. Gain-of-function genetic models by TSC1 deletion in HSCs or osteoclast precursors cause constitutive mTORC1 activation, impairing osteoclastogenesis. Pharmacologically, rapamycin treatment at low but clinically relevant doses exacerbates osteoclast differentiation and bone resorption, leading to bone loss. Mechanistically, RANKL inactivates mTORC1 via calcineurin-mediated mTORC1 dephosphorylation, consequently activating NFATc1 by reducing mTORC1-mediated NFATc1 phosphorylation. These findings uncover biphasic roles of mTORC1 in osteoclastogenesis, dosage-dependent effects of rapamycin on bone, and a previously unrecognized calcineurin-mTORC1-NFATc1 phosphorylation-regulatory signaling cascade.

Milk lipid regulation at the maternal-offspring interface.

Milk lipids provide a large proportion of energy, nutrients, essential fatty acids, and signaling molecules for the newborns, the synthesis of which is a tightly controlled process. Dysregulated milk lipid production and composition may be detrimental to the growth, development, health and survival of the newborns. Many genetically modified animal models have contributed to our understanding of milk lipid regulation in the lactating mammary gland. In this review, we discuss recent advances in our knowledge of the mechanisms that control milk lipid biosynthesis and secretion during lactation, and how maternal genetic and dietary defects impact milk lipid composition and consequently offspring traits.