A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition.

Cholecystokinin (CCK) enables excitatory circuit long-term potentiation (LTP). Here, we investigated its involvement in the enhancement of inhibitory synapses. Activation of GABA neurons suppressed neuronal responses in the neocortex to a forthcoming auditory stimulus in mice of both sexes. High-frequency laser stimulation (HFLS) of GABAergic neurons potentiated this suppression. HFLS of CCK interneurons could induce the LTP of their inhibition toward pyramidal neurons. This potentiation was abolished in CCK knock-out mice but intact in mice with both CCK1R and 2R knockout of both sexes. Next, we combined bioinformatics analysis, multiple unbiased cell-based assays, and histology examinations to identify a novel CCK receptor, GPR173. We propose GPR173 as CCK3R, which mediates the relationship between cortical CCK interneuron signaling and inhibitory LTP in the mice of either sex. Thus, GPR173 might represent a promising therapeutic target for brain disorders related to excitation and inhibition imbalance in the cortex.SIGNIFICANCE STATEMENT CCK, the most abundant and widely distributed neuropeptide in the CNS, colocalizes with many neurotransmitters and modulators. GABA is one of the important inhibitory neurotransmitters, and much evidence shows that CCK may be involved in modulating GABA signaling in many brain areas. However, the role of CCK-GABA neurons in the cortical microcircuits is still unclear. We identified a novel CCK receptor, GPR173, localized in the CCK-GABA synapses and mediated the enhancement of the GABA inhibition effect, which might represent a promising therapeutic target for brain disorders related to excitation and inhibition imbalance in the cortex.

Exclusion of HDAC1/2 complexes by oncogenic nuclear condensates.

Nuclear condensates have been shown to regulate cell fate control, but its role in oncogenic transformation remains largely unknown. Here we show acquisition of oncogenic potential by nuclear condensate remodeling. The proto-oncogene SS18 and its oncogenic fusion SS18-SSX1 can both form condensates, but with drastically different properties and impact on 3D genome architecture. The oncogenic condensates, not wild type ones, readily exclude HDAC1 and 2 complexes, thus, allowing aberrant accumulation of H3K27ac on chromatin loci, leading to oncogenic expression of key target genes. These results provide the first case for condensate remodeling as a transforming event to generate oncogene and such condensates can be targeted for therapy. One sentence summary: Expulsion of HDACs complexes leads to oncogenic transformation.

Long Non-Coding RNA Malat1 Increases the Rescuing Effect of Quercetin on TNFα-Impaired Bone Marrow Stem Cell Osteogenesis and Ovariectomy-Induced Osteoporosis.

Osteoporosis, a common systematic bone homeostasis disorder related disease, still urgently needs innovative treatment methods. Several natural small molecules were found to be effective therapeutics in osteoporosis. In the present study, quercetin was screened out from a library of natural small molecular compounds by a dual luciferase reporter system. Quercetin was found to upregulate Wnt/ß-catenin while inhibiting NF-κB signaling activities, and thereby rescuing osteoporosis-induced tumor necrosis factor alpha (TNFα) impaired BMSCs osteogenesis. Furthermore, a putative functional lncRNA, Malat1, was shown to be a key mediator in quercetin regulated signaling activities and TNFα-impaired BMSCs osteogenesis, as mentioned above. In an ovariectomy (OVX)-induced osteoporosis mouse model, quercetin administration could significantly rescue OVX-induced bone loss and structure deterioration. Serum levels of Malat1 were also obviously rescued in the OVX model after quercetin treatment. In conclusion, our study demonstrated that quercetin could rescue TNFα-impaired BMSCs osteogenesis in vitro and osteoporosis-induced bone loss in vivo, in a Malat1-dependent manner, suggesting that quercetin may serve as a therapeutic candidate for osteoporosis treatment.

Design and biological evaluation of substituted 5,7-dihydro-6H-indolo[2,3-c]quinolin-6-one as novel selective Haspin inhibitors.

A library of substituted indolo[2,3-c]quinolone-6-ones was developed as simplified Lamellarin isosters. Synthesis was achieved from indole after a four-step pathway sequence involving iodination, a Suzuki-Miyaura cross-coupling reaction, and a reduction/lactamization sequence. The inhibitory activity of the 22 novel derivatives was assessed on Haspin kinase. Two of them possessed an IC50 of 1 and 2 nM with selectivity towards a panel of 10 other kinases including the parent kinases DYRK1A and CLK1. The most selective compound exerted additionally a very interesting cell effect on the osteosarcoma U-2 OS cell line.

Cholecystokinin release triggered by NMDA receptors produces LTP and sound-sound associative memory.

Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we show that a cholecystokinin (CCK)-B receptor (CCKBR) antagonist blocks high-frequency stimulation-induced neocortical LTP, whereas local infusion of CCK induces LTP. CCK-/- mice lacked neocortical LTP and showed deficits in a cue-cue associative learning paradigm; and administration of CCK rescued associative learning deficits. High-frequency stimulation-induced neocortical LTP was completely blocked by either the NMDAR antagonist or the CCKBR antagonist, while application of either NMDA or CCK induced LTP after low-frequency stimulation. In the presence of CCK, LTP was still induced even after blockade of NMDARs. Local application of NMDA induced the release of CCK in the neocortex. These findings suggest that NMDARs control the release of CCK, which enables neocortical LTP and the formation of cue-cue associative memory.

The "elusive DMOAD": Aggrecanase inhibition from laboratory to clinic.

From the time of their discovery in 1999, the aggrecanases, and ADAMTS-5 in particular, have been heavily investigated as targets for disease-modifying osteoarthritis drug (DMOAD) development. Here, we provide a brief narrative review of the discovery efforts to target these enzymes, and how this led to the current ongoing programmes that hold promise for the future. We discuss a comparison of inhibition of collagen breakdown versus inhibition of aggrecan breakdown. We then summarise existing programmes that target ADAMTS-5, including small molecule inhibitors, monoclonal neutralising antibodies and nanobodies, and gene editing technologies. We also briefly discuss the potential analgesic effects this strategy may offer in addition to its joint-protective effects.

Evaluation of spiropiperidine hydantoins as a novel class of antimalarial agents.

Given the rise of parasite resistance to all currently used antimalarial drugs, the identification of novel chemotypes with unique mechanisms of action is of paramount importance. Since Plasmodium expresses a number of aspartic proteases necessary for its survival, we have mined antimalarial datasets for drug-like aspartic protease inhibitors. This effort led to the identification of spiropiperidine hydantoins, bearing similarity to known inhibitors of the human aspartic protease ß-secretase (BACE), as new leads for antimalarial drug discovery. Spiropiperidine hydantoins have a dynamic structure-activity relationship profile with positions identified as being tolerant of a variety of substitution patterns as well as a key piperidine N-benzyl phenol pharmacophore. Lead compounds 4e (CWHM-123) and 12k (CWHM-505) are potent antimalarials with IC50 values against Plasmodium falciparum 3D7 of 0.310 µM and 0.099 µM, respectively, and the former features equivalent potency on the chloroquine-resistant Dd2 strain. Remarkably, these compounds do not inhibit human aspartic proteases BACE, cathepsins D and E, or Plasmodium plasmepsins II and IV despite their similarity to known BACE inhibitors. Although the current leads suffer from poor metabolic stability, they do fit into a drug-like chemical property space and provide a new class of potent antimalarial agents for further study.

Cholecystokinin B receptor agonists alleviates anterograde amnesia in cholecystokinin-deficient and aged Alzheimer's disease mice.

BACKGROUND: As one major symptom of Alzheimer's disease (AD), anterograde amnesia describes patients with an inability in new memory formation. The crucial role of the entorhinal cortex in forming new memories has been well established, and the neuropeptide cholecystokinin (CCK) is reported to be released from the entorhinal cortex to enable neocortical associated memory and long-term potentiation. Though several studies reveal that the entorhinal cortex and CCK are related to AD, it is less well studied. It is unclear whether CCK is a good biomarker or further a great drug candidate for AD. METHODS: mRNA expressions of CCK and CCK-B receptor (CCKBR) were examined in two mouse models, 3xTg AD and CCK knock-out (CCK-/-) mice. Animals' cognition was investigated with Morris water maze, novel object recognition test and neuroplasticity with in-vitro electrophysiological recording. Drugs were given intraperitoneally to animals to investigate the rescue effects on cognitive deficits, or applied to brain slices directly to explore the influence in inducement of long-term potentiation. RESULTS: Aged 3xTg AD mice exhibited reduced CCK mRNA expression in the entorhinal cortex but reduced CCKBR expression in the neocortex and hippocampus, and impaired cognition and neuroplasticity comparable with CCK-/- mice. Importantly, the animals displayed improved performance and enhanced long-term potentiation after the treatment of CCKBR agonists. CONCLUSIONS: Here we provide more evidence to support the role of CCK in learning and memory and its potential to treat AD. We elaborated on the rescue effect of a promising novel drug, HT-267, on aged 3xTg AD mice. Although the physiological etiology of CCK in AD still needs to be further investigated, this study sheds light on a potential pharmaceutical candidate for AD and dementia.

Azetidines Kill Multidrug-Resistant Mycobacterium tuberculosis without Detectable Resistance by Blocking Mycolate Assembly.

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.

Rescue of ATP7B function in hepatocyte-like cells from Wilson's disease induced pluripotent stem cells using gene therapy or the chaperone drug curcumin.

Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a unique platform for modeling liver genetic diseases and performing drug-toxicity screening in vitro. Wilson's disease is a genetic disease caused by mutations in the ATP7B gene, whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. Interestingly, the spectrum of ATP7B mutations is vast and can influence clinical presentation (a variable spectrum of hepatic and neural manifestations), though the reason is not well understood. We describe the generation of iPSCs from a Chinese patient with Wilson's disease that bears the R778L Chinese hotspot mutation in the ATP7B gene. These iPSCs were pluripotent and could be readily differentiated into hepatocyte-like cells that displayed abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Moreover, gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B or treatment with the chaperone drug curcumin could reverse the functional defect in vitro. Hence, our work describes an attractive model for studying the pathogenesis of Wilson's disease that is valuable for screening compounds or gene therapy approaches aimed to correct the abnormality. In the future, once relevant safety concerns (including the stability of the mature liver-like phenotype) and technical issues for the transplantation procedure are solved, hepatocyte-like cells from similarly genetically corrected iPSCs could be an option for autologous transplantation in Wilson's disease.

Nanoparticles for improved local retention after intra-articular injection into the knee joint.

PURPOSE: To evaluate using cationic polymeric nanoparticles that interact with hyaluronate to form ionically cross-linked hydrogels to increase the intra-articular retention time of osteoarthritis drugs in the synovial cavity. METHODS: In vitro tests included nanoparticle release from cross-linked hydrogels using syringe and membrane dissolution tests, viscosity measurement of synovial fluid containing hydrogels, and release-rate measurement for a model active conjugated to a cationically substituted dextran using a hydrolyzable ester linkage in a sink dissolution test. Nanoparticle retention after intra-articular injection into rat knees was measured in vivo using fluorescence molecular tomography. RESULTS: Diffusional and convective transport of cationic nanoparticles from ionically cross-linked hydrogels formed in synovial fluid was slower in vitro than for uncharged nanoparticles. Hydrogels formed after the nanoparticles were mixed with synovial fluid did not appreciably alter the viscosity of the synovial fluid in vitro. In vitro release of a conjugated peptide from the cationic nanoparticles was approximately 20% per week. After intra-articular injection in rat knees, 70% of the nanoparticles were retained in the joint for 1 week. CONCLUSIONS: This study demonstrates the feasibility of using cationic polymeric nanoparticles to increase the retention of therapeutic agents in articular joints for indications such as osteoarthritis.

Genetically Engineered Mouse Models Reveal the Importance of Proteases as Osteoarthritis Drug Targets.

More than two decades of research has revealed a combination of proteases that determine cartilage degradation in osteoarthritis. These include metalloproteinases, which degrade the major macromolecules in cartilage, aggrecan and type II collagen, serine proteases, and cysteine proteases, for example cathepsin K. This review summarizes the function of proteases in osteoarthritis progression, as revealed by studies of genetically engineered mouse models. A brief overview of the biochemical characteristics and features of several important proteases is provided, with the objective of increasing understanding of their function. Published data reveal at least three enzymes to be major targets for osteoarthritis drug development: ADAMTS-5, MMP-13, and cathepsin K. In surgical models of osteoarthritis, mice lacking these enzymes are protected from cartilage damage and, to varying degrees, from bone changes. In-vivo studies targeting these proteases with selective small-molecule inhibitors have been performed for a variety of animal models. Mouse models will provide opportunities for future tests of the therapeutic effect of protease inhibitors, both on progression of structural damage to the joint and on associated pain.

Cartilage organoids for cartilage development and cartilage-associated disease modeling.

Cartilage organoids have emerged as powerful modelling technology for recapitulation of joint embryonic events, and cartilage regeneration, as well as pathophysiology of cartilage-associated diseases. Recent breakthroughs have uncovered "mini-joint" models comprising of multicellular components and extracellular matrices of joint cartilage for development of novel disease-modifying strategies for personalized therapeutics of cartilage-associated diseases. Here, we hypothesized that LGR5-expressing embryonic joint chondroprogenitor cells are ideal stem cells for the generation of cartilage organoids as "mini-joints" ex vivo "in a dish" for embryonic joint development, cartilage repair, and cartilage-associated disease modelling as essential research models of drug screening for further personalized regenerative therapy. The pilot research data suggested that LGR5-GFP-expressing embryonic joint progenitor cells are promising for generation of cartilage organoids through gel embedding method, which may exert various preclinical and clinical applications for realization of personalized regenerative therapy in the future.

Establishing extended pluripotent stem cells from human urine cells.

BACKGROUND: Extended pluripotent stem cells (EPSCs) can contribute to both embryonic and trophectoderm-derived extraembryonic tissues. Therefore, EPSCs have great application significance for both research and industry. However, generating EPSCs from human somatic cells remains inefficient and cumbersome. RESULTS: In this study, we established a novel and robust EPSCs culture medium OCM175 with defined and optimized ingredients. Our OCM175 medium contains optimized concentration of L-selenium-methylcysteine as a source of selenium and ROCK inhibitors to maintain the single cell passaging ability of pluripotent stem cells. We also used Matrigel or the combination of laminin 511 and laminin 521(1:1) to bypass the requirement of feeder cells. With OCM175 medium, we successfully converted integration-free iPSCs from easily available human Urine-Derived Cells (hUC-iPSCs) into EPSCs (O-IPSCs). We showed that our O-IPSCs have the ability to form both intra- and extra- embryonic chimerism, and could contribute to the trophoblast ectoderm lineage and three germ layer cell lineages. CONCLUSIONS: In conclusion, our novel OCM175 culture medium has defined, optimized ingredients, which enables efficient generation of EPSCs in a feeder free manner. With the robust chimeric and differentiation potential, we believe that this system provides a solid basis to improve the application of EPSCs in regenerative medicine.

O-alg-THAM/gel hydrogels functionalized with engineered microspheres based on mesenchymal stem cell secretion recruit endogenous stem cells for cartilage repair.

Lacking self-repair abilities, injuries to articular cartilage can lead to cartilage degeneration and ultimately result in osteoarthritis. Tissue engineering based on functional bioactive scaffolds are emerging as promising approaches for articular cartilage regeneration and repair. Although the use of cell-laden scaffolds prior to implantation can regenerate and repair cartilage lesions to some extent, these approaches are still restricted by limited cell sources, excessive costs, risks of disease transmission and complex manufacturing practices. Acellular approaches through the recruitment of endogenous cells offer great promise for in situ articular cartilage regeneration. In this study, we propose an endogenous stem cell recruitment strategy for cartilage repair. Based on an injectable, adhesive and self-healable o-alg-THAM/gel hydrogel system as scaffolds and a biophysio-enhanced bioactive microspheres engineered based on hBMSCs secretion during chondrogenic differentiation as bioactive supplement, the as proposed functional material effectively and specifically recruit endogenous stem cells for cartilage repair, providing new insights into in situ articular cartilage regeneration.

Entorhinohippocampal cholecystokinin modulates spatial learning by facilitating neuroplasticity of hippocampal CA3-CA1 synapses.

The hippocampus is broadly impacted by neuromodulations. However, how neuropeptides shape the function of the hippocampus and the related spatial learning and memory remains unclear. Here, we discover the crucial role of cholecystokinin (CCK) in heterosynaptic neuromodulation from the medial entorhinal cortex (MEC) to the hippocampus. Systematic knockout of the CCK gene impairs CA3-CA1 LTP and space-related performance. The MEC provides most of the CCK-positive neurons projecting to the hippocampal region, which potentiates CA3-CA1 long-term plasticity heterosynaptically in a frequency- and NMDA receptor (NMDAR)-dependent manner. Selective inhibition of MEC CCKergic neurons or downregulation of their CCK mRNA levels also impairs CA3-CA1 LTP formation and animals' performance in the water maze. This excitatory extrahippocampal projection releases CCK upon high-frequency excitation and is active during animal exploration. Our results reveal the critical role of entorhinal CCKergic projections in bridging intra- and extrahippocampal circuitry at electrophysiological and behavioral levels.

Human fetal mesenchymal stem cells secretome promotes scarless diabetic wound healing through heat-shock protein family.

The high mortality rate of patients with diabetic foot ulcers is urging the appearance of an effective biomedical drug. Senescence is one of the major reasons of aging-induced decline in the diabetic wound. Our previous studies have demonstrated the anti-senescence effect of secretomes derived from human fetal mesenchymal stem cells (hfMSC). The present study tends to explore the potential role of hfMSC secretome (HFS) in wound healing through anti-aging. Meanwhile, we try to overcome several obstacles in the clinical application of stem cell secretome. A verticle bioreactor and microcarriers are employed to expand hfMSC and produce the HFS on a large scale. The HFS was then subjected to lyophilization (L-HFS). The PLGA (poly lactic-co-glycolic acid) particles were used to encapsulate and protect L-HFS from degradation in the streptozotocin (STZ)-induced diabetic rat model. Results showed that HFS-PLGA significantly enhanced wound healing by promoting vascularization and inhibiting inflammation in the skin wound bed. We further analyzed the contents of HFS. Isobaric tag for relative and absolute quantitation (ITRAQ) and label-free methods were used to identify peptides in the secretome. Bioinformatics analysis indicated that exosome production-related singling pathways and heat-shock protein family could be used as bio-functional markers and quality control for stem cell secretome production.

Low-Dose Staphylococcal Enterotoxin C2 Mutant Maintains Bone Homeostasis via Regulating Crosstalk between Bone Formation and Host T-Cell Effector Immunity.

Studies in recent years have highlighted an elaborate crosstalk between T cells and bone cells, suggesting that T cells may be alternative therapeutic targets for the maintenance of bone homeostasis. Here, it is reported that systemic administration of low-dose staphylococcal enterotoxin C2 (SEC2) 2M-118, a form of mutant superantigen, dramatically alleviates ovariectomy (OVX)-induced bone loss via modulating T cells. Specially, SEC2 2M-118 treatment increases trabecular bone mass significantly via promoting bone formation in OVX mice. These beneficial effects are largely diminished in T-cell-deficient nude mice and can be rescued by T-cell reconstruction. Neutralizing assays determine interferon gamma (IFN-γ) as the key factor that mediates the beneficial effects of SEC2 2M-118 on bone. Mechanistic studies demonstrate that IFN-γ stimulates Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling, leading to enhanced production of nitric oxide, which further activates p38 mitogen-activated protein kinase (MAPK) and Runt-related transcription factor 2 (Runx2) signaling and promotes osteogenic differentiation. IFN-γ also directly inhibits osteoclast differentiation, but this effect is counteracted by proabsorptive factors tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß) secreted from IFN-γ-stimulated macrophages. Taken together, this work provides clues for developing innovative approaches which target T cells for the prevention and treatment of osteoporosis.

A role for PACE4 in osteoarthritis pain: evidence from human genetic association and null mutant phenotype.

OBJECTIVES: The aim of this study was to assess if genetic variation in the PACE4 (paired amino acid converting enzyme 4) gene Pcsk6 influences the risk for symptomatic knee osteoarthritis (OA). METHODS: Ten PCSK6 single nucleotide polymorphisms were tested for association in a discovery cohort of radiographic knee OA (n=156 asymptomatic and 600 symptomatic cases). Meta-analysis of the minor allele at rs900414 was performed in three additional independent cohorts (total n=674 asymptomatic and 2068 symptomatic). Pcsk6 knockout mice and wild-type C57BL/6 mice were compared in a battery of algesiometric assays, including hypersensitivity in response to intraplantar substance P, pain behaviours in response to intrathecal substance P and pain behaviour in the abdominal constriction test. RESULTS: In the discovery cohort of radiographic knee OA, an intronic single nucleotide polymorphism at rs900414 was significantly associated with symptomatic OA. Replication in three additional cohorts confirmed that the minor allele at rs900414 was consistently increased among asymptomatic compared to symptomatic radiographic knee OA cases in all four cohorts. A fixed-effects meta-analysis yielded an OR=1.35 (95% CI 1.17 to 1.56; p=4.3×10(-5) and no significant between-study heterogeneity). Studies in mice revealed that Pcsk6 knockout mice were significantly protected against pain in a battery of algesiometric assays. CONCLUSIONS: These results suggest that a variant in PCSK6 is strongly associated with protection against pain in knee OA, offering some insight as to why, in the presence of the same structural damage, some individuals develop chronic pain and others are protected. Studies in Pcsk6 null mutant mice further implicate PACE4 in pain.

Molecular design of dual inhibitors of PI3K and potential molecular target of cancer for its treatment: A review.

Aberrant activation of the phosphoinositide 3-kinase (PI3K) signaling network is a key event in many human cancers and therefore enormous efforts have been made in the development of PI3K inhibitors. However, due to intrinsic and acquired resistance as well as poor drug tolerance, limited therapeutic efficacy has been achieved with these agents. In view of the fact that PI3K inhibitors can show synergistic antitumor effects with other cancer agents, namely mammalian target of rapamycin (mTOR) inhibitors, histone deacetylase (HDAC) inhibitors and mitogen-activated protein kinase (MEK) inhibitors, dual inhibition of both targets by a single-molecule is regarded as a promising complementary or alternative therapeutic strategy to overcome the drawbacks of just PI3K monotherapy. In this review, we discuss the theoretical foundation for designing PI3K-based dual-target inhibitors and summarize the structure-activity relationships and clinical progress of these dual-binding agents.