Host immunomodulatory lipids created by symbionts from dietary amino acids.

Small molecules derived from symbiotic microbiota critically contribute to intestinal immune maturation and regulation1. However, little is known about the molecular mechanisms that control immune development in the host-microbiota environment. Here, using a targeted lipidomic analysis and synthetic approach, we carried out a multifaceted investigation of immunomodulatory α-galactosylceramides from the human symbiont Bacteroides fragilis (BfaGCs). The characteristic terminal branching of BfaGCs is the result of incorporation of branched-chain amino acids taken up in the host gut by B. fragilis. A B. fragilis knockout strain that cannot metabolize branched-chain amino acids showed reduced branching in BfaGCs, and mice monocolonized with this mutant strain had impaired colonic natural killer T (NKT) cell regulation, implying structure-specific immunomodulatory activity. The sphinganine chain branching of BfaGCs is a critical determinant of NKT cell activation, which induces specific immunomodulatory gene expression signatures and effector functions. Co-crystal structure and affinity analyses of CD1d-BfaGC-NKT cell receptor complexes confirmed the interaction of BfaGCs as CD1d-restricted ligands. We present a structural and molecular-level paradigm of immunomodulatory control by interactions of endobiotic metabolites with diet, microbiota and the immune system.

IL-27-producing B-1a cells suppress neuroinflammation and CNS autoimmune diseases.

Regulatory B cells (Breg cells) that secrete IL-10 or IL-35 (i35-Breg) play key roles in regulating immunity in tumor microenvironment or during autoimmune and infectious diseases. Thus, loss of Breg function is implicated in development of autoimmune diseases while aberrant elevation of Breg prevents sterilizing immunity, exacerbates infectious diseases, and promotes cancer metastasis. Breg cells identified thus far are largely antigen-specific and derive mainly from B2-lymphocyte lineage. Here, we describe an innate-like IL-27-producing natural regulatory B-1a cell (i27-Breg) in peritoneal cavity and human umbilical cord blood. i27-Bregs accumulate in CNS and lymphoid tissues during neuroinflammation and confers protection against CNS autoimmune disease. i27-Breg immunotherapy ameliorated encephalomyelitis and uveitis through up-regulation of inhibitory receptors (Lag3, PD-1), suppression of Th17/Th1 responses, and propagating inhibitory signals that convert conventional B cells to regulatory lymphocytes that secrete IL-10 and/or IL-35 in eye, brain, or spinal cord. Furthermore, i27-Breg proliferates in vivo and sustains IL-27 secretion in CNS and lymphoid tissues, a therapeutic advantage over administering biologics (IL-10, IL-35) that are rapidly cleared in vivo. Mutant mice lacking irf4 in B cells exhibit exaggerated increase of i27-Bregs with few i35-Bregs, while mice with loss of irf8 in B cells have abundance of i35-Bregs but defective in generating i27-Bregs, identifying IRF8/BATF and IRF4/BATF axis in skewing B cell differentiation toward i27-Breg and i35-Breg developmental programs, respectively. Consistent with its developmental origin, disease suppression by innate i27-Bregs is neither antigen-specific nor disease-specific, suggesting that i27-Breg would be effective immunotherapy for a wide spectrum of autoimmune diseases.

Inflachromene attenuates seizure severity in mouse epilepsy models via inhibiting HMGB1 translocation.

Epilepsy is not well controlled by current anti-seizure drugs (ASDs). High mobility group box 1 (HMGB1) is a DNA-binding protein in the nucleus regulating transcriptional activity and maintaining chromatin structure and DNA repair. In epileptic brains, HMGB1 is released by activated glia and neurons, interacting with various receptors like Toll-like receptor 4 (TLR4) and downstream glutamatergic NMDA receptor, thus enhancing neural excitability. But there is a lack of small-molecule drugs targeting the HMGB1-related pathways. In this study we evaluated the therapeutic potential of inflachromene (ICM), an HMGB-targeting small-molecule inhibitor, in mouse epilepsy models. Pentylenetetrazol-, kainic acid- and kindling-induced epilepsy models were established in mice. The mice were pre-treated with ICM (3, 10 mg/kg, i.p.). We showed that ICM pretreatment significantly reduced the severity of epileptic seizures in all the three epilepsy models. ICM (10 mg/kg) exerted the most apparent anti-seizure effect in kainic acid-induced epileptic status (SE) model. By immunohistochemical analysis of brain sections from kainic acid-induced SE mice, we found that kainic acid greatly enhanced HMGB1 translocation in the hippocampus, which was attenuated by ICM pretreatment in subregion- and cell type-dependent manners. Notably, in CA1 region, the seizure focus, ICM pretreatment mainly inhibited HMGB1 translocation in microglia. Furthermore, the anti-seizure effect of ICM was related to HMGB1 targeting, as pre-injection of anti-HMGB1 monoclonal antibody (5 mg/kg, i.p.) blocked the seizure-suppressing effect of ICM in kainic acid-induced SE model. In addition, ICM pretreatment significantly alleviated pyramidal neuronal loss and granule cell dispersion in kainic acid-induced SE model. These results demonstrate that ICM is an HMGB-targeting small molecule with anti-seizure potential, which may help develop a potential drug for treating epilepsy.

Ultrafluorogenic Monochromophore-Type BODIPY-Tetrazine Series for Dual-Color Bioorthogonal Imaging with a Single Probe.

Various fluorogenic probes utilizing tetrazine (Tz) as a fluorescence quencher and bioorthogonal reaction partner have been extensively studied over the past few decades. Herein, we synthesized a series of boron-dipyrromethene (BODIPY)-Tz probes using monochromophoric design strategy for bioorthogonal cellular imaging. The BODIPY-Tz probes exhibited excellent bicyclo[6.1.0]nonyne (BCN)-selective fluorogenicity with three- to four-digit-fold enhancements in fluorescence over a wide range of emission wavelengths, including the far-red region. Furthermore, we demonstrated the applicability of BODIPY-Tz probes in bioorthogonal fluorescence imaging of cellular organelles without washing steps. We also elucidated the aromatized pyridazine moiety as the origin of BCN-selective fluorogenic behavior. Additionally, we discovered that the fluorescence of the trans-cyclooctene (TCO) adducts was quenched in aqueous media via photoinduced electron transfer (PeT) process. Interestingly, we observed a distinctive recovery of the initially quenched fluorescence of BODIPY-Tz-TCO upon exposure to hydrophobic media, accompanied by a significant bathochromic shift of its emission wavelength relative to that exhibited by the corresponding BODIPY-Tz-BCN. Leveraging this finding, for the first time, we achieved dual-color bioorthogonal cellular imaging with a single BODIPY-Tz probe.

Targeted Protein Upregulation of STING for Boosting the Efficacy of Immunotherapy.

Modulating target proteins via the ubiquitin-proteasome system has recently expanded the scope of pharmacological inventions. Stimulator of interferon genes (STING) is an auspicious target for immunotherapy. Seminal studies envisioned the importance of STING as well as the utility of its agonists in immunotherapy outcomes. Herein, we suggest UPPRIS (upregulation of target proteins by protein-protein interaction strategy) to pharmacologically increase cellular STING levels for improved immunotherapy. We discovered the small molecule SB24011 that inhibits STING-TRIM29 E3 ligase interaction, thus blocking TRIM29-induced degradation of STING. SB24011 enhanced STING immunity by upregulating STING protein levels, which robustly potentiated the immunotherapy efficacy of STING agonist and anti-PD-1 antibody via systemic anticancer immunity. Overall, we demonstrated that targeted protein upregulation of STING can be a promising approach for immuno-oncology.

Modeling PNPLA3-Associated NAFLD Using Human-Induced Pluripotent Stem Cells.

BACKGROUND AND AIMS: NAFLD is a growing public health burden. However, the pathogenesis of NAFLD has not yet been fully elucidated, and the importance of genetic factors has only recently been appreciated. Genomic studies have revealed a strong association between NAFLD progression and the I148M variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3). Nonetheless, very little is known about the mechanisms by which this gene and its variants can influence disease development. To investigate these mechanisms, we have developed an in vitro model that takes advantage of the unique properties of human-induced pluripotent stem cells (hiPSCs) and the CRISPR/CAS9 gene editing technology. APPROACH AND RESULTS: We used isogenic hiPSC lines with either a knockout (PNPLA3KO ) of the PNPLA3 gene or with the I148M variant (PNPLA3I148M ) to model PNPLA3-associated NAFLD. The resulting hiPSCs were differentiated into hepatocytes, treated with either unsaturated or saturated free fatty acids to induce NAFLD-like phenotypes, and characterized by various functional, transcriptomic, and lipidomic assays. PNPLA3KO hepatocytes showed higher lipid accumulation as well as an altered pattern of response to lipid-induced stress. Interestingly, loss of PNPLA3 also caused a reduction in xenobiotic metabolism and predisposed PNPLA3KO cells to be more susceptible to ethanol-induced and methotrexate-induced toxicity. The PNPLA3I148M cells exhibited an intermediate phenotype between the wild-type and PNPLA3KO cells. CONCLUSIONS: Together, these results indicate that the I148M variant induces a loss of function predisposing to steatosis and increased susceptibility to hepatotoxins.

Fluorophore Labeling of Proteins: a Versatile Trigger-Release-Conjugation Platform Based on the Quinone Methide Chemistry.

In situ conjugation of fluorescent molecules to biomolecules such as proteins under spatiotemporal control offers a powerful means for studying biological systems. For that purpose, the o-quinone methide chemistry involving a sequence of the trigger-release-conjugation (TRC) process provides a versatile conjugation method. We have developed a new TRC platform bearing a quaternary ammonium salt for the release process, which can be structurally modified and readily synthesized from commonly used aryl alcohol-type organic fluorophores under environmentally benign conditions. We show that different aryl alcohol fluorophores containing the o-(morpholinium)methyl group for the release process allow efficient fluorophore labeling of proteins under both light- and chemical-triggering conditions. The bioconjugation in cells as well as in tissues was further demonstrated with an o-(morpholinium)methyl analogue containing a triggering group sensitive to reactive oxygen species. The new TRC system thus provides a versatile and unique platform for in situ fluorophore labeling of proteins in biological systems under spatiotemporal control.

Systematic Exploration of Furoindolizine-Based Molecular Frameworks towards a Versatile Fluorescent Platform.

The photophysical behaviors of fluorescent molecules largely determine their major utility in biological studies. Despite their well-defined characteristics, classical fluorophores have often been challenged by their limited synthetic methodology and tunability in adjusting intrinsic optical properties. A novel heterocyclic core equipped with modular functional groups could offer the flexibility to control its photophysical properties with a minimum synthetic effort. By conducting a systematic analysis guided by quantum calculations, we proposed the furoindolizine-based molecular framework as a unique fluorescent platform capable of providing versatile photophysical properties with minimal structural modification. A broad tunability of furoindolizine derivatives' photophysical properties such as emission wavelength, Stokes shift, fluorescent brightness, and charge transfer characteristics was achieved through synergistic interaction between two functional moieties. Furthermore, this modular platform led to live-cell imaging probes with two distinct optical features simply by reorganizing a pair of functional moieties.

Inhibition of ACE2-Spike Interaction by an ACE2 Binder Suppresses SARS-CoV-2 Entry.

The emergence of SARS-CoV-2 variants is a significant concern in developing effective therapeutics and vaccines in the middle of the ongoing COVID-19 pandemic. Here, we have identified a novel small molecule that inhibited the interactions between SARS-CoV-2 spike RBDs and ACE2 by modulating ACE2 without impairing its enzymatic activity necessary for normal physiological functions. Furthermore, the identified compounds suppressed viral infection in cultured cells by inhibiting the entry of ancestral and variant SARS-CoV-2. Our study suggests that targeting ACE2 could be a novel therapeutic strategy to inhibit SARS-CoV-2 entry into host cells and prevent the development of COVID-19.

The blockade of cytoplasmic HMGB1 modulates the autophagy/apoptosis checkpoint in stressed islet beta cells.

High mobility group (HMGB1) is an alarmin known to be harmful to pancreatic beta cells and associated with diabetes mellitus pathogenesis and pancreatic islet graft failure. It has been long thought that the suppression of HMGB1 molecule is beneficial to the beta cells. However, recent studies have indicated that cytoplasmic HMGB1 (cHMGB1) could function as a modulator to relieve cells from apoptotic stress by autophagy induction. Particularly, pancreatic beta cells have been known to utilize the autophagy-to-apoptosis switch when exposed to hypoxia or lipotoxicity. This study aimed to investigate the beta cells under hypoxic and lipotoxic stress while utilizing a small molecule inhibitor of HMGB1, inflachromene (ICM) which can suppress cHMGB1 accumulation. It was revealed that under cellular stress, blockade of cHMGB1 accumulation decreased the viability of islet grafts, primary islets and MIN6 cells. MIN6 cells under cHMGB1 blockade along with lipotoxic stress showed decreased autophagic flux and increased apoptosis. Moreover, cHMGB1 blockade in HFD-fed mice produced unfavorable outcomes on their glucose tolerance. In sum, these results suggested the role of cHMGB1 within beta cell autophagy/apoptosis checkpoint. Given the importance of autophagy in beta cells under apoptotic stresses, this study might provide further insights regarding HMGB1 and diabetes.

Phenotypic Discovery of Neuroprotective Agents by Regulation of Tau Proteostasis via Stress-Responsive Activation of PERK Signaling.

Tau protein aggregates are a recognized neuropathological feature in Alzheimer's disease as well as many other neurodegenerative disorders, known as tauopathies. The development of tau-targeting therapies is therefore extremely important but efficient strategies or protein targets are still unclear. Here, we performed a cell-based phenotypic screening under endoplasmic reticulum (ER) stress conditions and identified a small molecule, SB1617, capable of suppressing abnormal tau protein aggregation. By applying label-free target identification technology, we revealed that the transient enhancement of protein kinase-like endoplasmic reticulum kinase (PERK) signaling pathway through the inhibition of stress-responsive SB1617 targets, PDIA3 and DNAJC3, is an effective strategy for regulating proteostasis in tauopathies. The molecular mechanism and the promising efficacy of SB1617 were demonstrated in neuronal cells and a mouse model with traumatic brain injury, a tauopathy known to involve ER stress.

Discovery of Small-Molecule Modulators of Protein-RNA Interactions by Fluorescence Intensity-Based Binding Assay.

Protein-RNA interactions mediate various cellular processes, the dysregulation of which has been associated with a list of diseases. Thus, novel experimental tools for monitoring protein-RNA interactions are highly desirable to identify new chemical modulators of these therapeutic targets. In this study, we constructed simple fluorescence intensity-based protein-RNA binding assays by testing multiple environment-sensitive organic fluorophores. We selected the oncogenic interaction between Lin28 and the let-7 microRNA and the important immunomodulatory Roquin-Tnf CDE interaction as representative targets. We adapted this assay to high-throughput screening for the identification of pyrazolyl thiazolidinedione-type molecules as potent small-molecule inhibitors of protein-microRNA interactions. We clearly showed the structure-activity relationships of this new class of Lin28-let-7 interaction inhibitors, and confirmed that cellular mature let-7 microRNAs and their target genes could be modulated upon treatment with the pyrazolyl thiazolidinedione-type inhibitor. We expect that our simple and adaptable screening approach can be applied for the development of various assay systems aimed at the identification of bioactive small molecules targeting protein-RNA interactions.

Two-Photon and Multicolor Fluorogenic Bioorthogonal Probes Based on Tetrazine-Conjugated Naphthalene Fluorophores.

Herein, we report the use of two-photon fluorogenic probes using tetrazine-based bioorthogonal reactions with multicolor emissions that cover nearly all of the visible region. New fluorogenic probes were designed based on donor-acceptor-type naphthalene structures conjugated with a fluorescence-quenching tetrazine moiety for turn-on properties in one- and two-photon fluorescence. Our fluorescent probes showed a moderate to good turn-on ratio after bioorthogonal inverse electron demand Diels-Alder cycloaddition with trans-cyclooctenol in one- and two-photon fluorescence. We successfully applied our probes to mitochondria- and lysosome-selective bioorthogonal imaging in live cells with one-/two-photon and one-photon microscopy, respectively.

A Nonparametric SVM-Based REM Recapitulation Assisted by Voluntary Sensing Participants under Smart Contracts on Blockchain.

This paper proposes a blockchain-based automated frequency coordination system (BAFCS) for secure and reliable spectrum sharing without causing any harmful interference to an existing system. For the exact assessment of whether the incumbent is interfered with by the spectrum sharer, the received signal strength (RSS) associated with the incumbent should be measured with sufficient accuracy at every location within the area of interest. However, since it requires brute force to carry out empirical measurements around an entire region, to lessen the burden, only the confined portion of the RSSs associated with the incumbent as a kind of primary user are observed and the omitted residuals are conventionally estimated by carrying out the well-known Kriging interpolation with regard to the geostatistical characteristics. This paper proposes a frequency coordination system capable of identifying whether a requested frequency band can be eligible for spectrum sharing while exchanging adequate information over blockchain network to confirm the usability. This paper proposes the Support Vector Machine (SVM)-based Kriging interpolation for recapitulating the radio environment map (REM) when only a fraction of the RSS measurements is acquired by the voluntary sensing participant (VSP). The nonparametric modeling approach for variograms proposed in this paper was determined to have a vital role in making a confident decision regarding spectrum sharing. The simulation result confirmed the effectiveness and the superiority of the proposed BAFCS with several affirmative features, such as enabling the consensus-based approval of spectrum sharing, the secure transaction of the information, and reliable assurance of no harmful interference.

TM6SF2 Promotes Lipidation and Secretion of Hepatitis C Virus in Infected Hepatocytes.

BACKGROUND & AIMS: Hepatitis C virus (HCV) co-opts the very-low-density lipoprotein pathway for morphogenesis, maturation, and secretion, and circulates as lipoviroparticles (LVPs). We investigated the functions and underlying mechanisms of the lipid-associated TM6SF2 protein in modulating LVP formation and the HCV life cycle. METHODS: We knocked down or overexpressed TM6SF2 in hepatic cells and examined HCV infection, measuring viral RNA and protein levels and infectious LVP titers. The density of secreted LVPs was evaluated by iodixanol gradient assay. We measured levels and patterns of TM6SF2 in liver biopsies from 73 patients with chronic hepatitis C, livers of HCV-infected humanized Alb-uPA/SCID/beige mice, and HCV-infected Huh7.5.1 cells. RESULTS: TM6SF2 knockdown in hepatocytes reduced viral RNA and infectious viral particle secretion without affecting HCV genome replication, translation, or assembly. Overexpression of TM6SF2 reduced intracellular levels of HCV RNA and infectious LVPs, and conversely increased their levels in the culture supernatants. In HCV-infected cells, TM6SF2 overexpression resulted in production of more infectious LVPs in the lower-density fractions of supernatant. HCV infection increased TM6SF2 expression in cultured cells, humanized livers of mice, and liver tissues of HCV patients. TM6SF2 messenger RNA levels correlated positively with HCV RNA levels in liver biopsies from patients. SREBF2 appears to mediate the ability of HCV to increase the expression of TM6SF2 in hepatic cells. CONCLUSIONS: In studies of cells, mice and human liver tissues, we found TM6SF2 is required for maturation, lipidation, and secretion of infectious LVPs. HCV, in turn, up-regulates expression of TM6SF2 to facilitate productive infection.

Retraction notice to "Polo-like kinase 2 gene expression is regulated by the orphan nuclear receptor estrogen receptor-related receptor gamma (ERRγ)" [Biochem. Biophys. Res. Commun. 362 (1) (2007) 107-113].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The authors have indicated that Fig. 1D data originated from another source not specified in the article. They also indicated image duplication in Fig. 1A and B. The authors of this article would like to apologize to all affected parties.

High mobility group box 1 secretion blockade results in the reduction of early pancreatic islet graft loss.

Pancreatic islet transplantation has been known as the best cure for patients suffering from severe type 1 diabetes mellitus (T1DM). Despite meaningful advances in human allogeneic islet transplantation field, significant amounts of islet loss in early post-transplantation periods is still a big concern for clinicians. One of the major factors determining the fate of the islets is the danger-associated molecular patterns (DAMPs) secreted by activated immune cells or islets themselves under hypoxic stress. High mobility group box 1 (HMGB1) protein is one of the best characterized DAMP molecules associated with islets. HMGB1 is known to be passively released by transplanted murine islet cells after taking damages from cytokines, reactive oxygen species, and other DAMPS, and the released HMGB1 harms neighboring islet cells by interacting with receptors expressed on murine islets such as toll-like receptor 2 (TLR2) and TLR4, thereby forming a vicious cycle. Here, we show that a small molecule inhibitor inflachromene (ICM) was capable of blocking the secretion of HMGB1 from murine islet cells during the normoxic and hypoxic post-isolation period. Notably, the treatment of ICM during the islet isolation process resulted in decreased HMGB1 levels during the subsequent cell culture. ICM's in vivo efficacy was evaluated in murine syngeneic islet transplantation model, and it significantly reduced the serum and graft level of HMGB1. Ultimately, the intraperitoneal administration of ICM prevented the loss of marginal-mass islet grafts and reversed the diabetes in mice.

Phenotype-based discovery of a HeLa-specific cytotoxic molecule that downregulates HPV-mediated signaling pathways via oxidative damage.

Selective bioactive compounds have emerged as major players in chemical biology for their potential in disrupting diverse biological pathways with minimal adverse effects. Using phenotypic screening, we identified an anti-cancer agent, SB2001, with a highly specific cytotoxicity toward HeLa human cervical cancer cells. The subsequent mechanistic study revealed that SB2001 induced apoptotic cell death through restoring p53 function and suppressed the human papillomavirus (HPV)-mediated oncoprotein signaling pathway via oxidative damage in HeLa cells. SB2001 also selectively induced HeLa-specific tumor regression without any adverse effects in an in vivo tumor xenograft model, demonstrating its potential as a promising chemical probe.

Monochromophoric Design Strategy for Tetrazine-Based Colorful Bioorthogonal Probes with a Single Fluorescent Core Skeleton.

Fluorogenic bioorthogonal probes are ideal for fluorescent imaging in live cell conditions. By taking advantage of the dual functionality of tetrazine (Tz), as a bioorthogonal reaction unit as well as a fluorescence quencher, a fluorophore-Tz conjugate (FLTz) has been utilized for fluorescent live cell imaging via inverse electron-demand Diels-Alder (iEDDA) type bioorthogonal reactions. However, most FLTz strategies rely on a donor-acceptor-type energy transfer mechanism, which limits red-shifting of probes' emission wavelength without deterioration of the fluorescent turn-on/off ratio. To address this constraint, herein we present a monochromophoric design strategy for making a series of FLTzs spanning a broad range of emission colors. For the systematic comparison of design strategies with minimized structural differences, we selected indolizine-based emission-tunable Seoul-Fluor (SF) as a model fluorophore system. As a result, by inducing strong electronic coupling between Tz and π-conjugation systems of an indolizine core, we efficiently quench the fluorescence of SF-tetrazine conjugates (SFTzs) and achieved more than 1000-fold enhancement in fluorescence after iEDDA reaction with trans-cyclooctene (TCO). Importantly, we were able to develop a series of colorful SFTzs with a similar turn-on/off ratio regardless of their emission wavelength. The applicability as bioorthogonal probes was demonstrated with fluorescence bioimaging of innate microtubule and mitochondria using docetaxel-TCO and triphenylphosphonium-TCO in live cells without washing steps. We believe this study could provide new insight for the reliable and generally applicable molecular design strategy to develop bioorthogonal fluorogenic probes having an excellent turn-on ratio, regardless of their emission wavelength.