Cholecystokinin receptor type A are involved in the circadian rhythm of the mouse retina.

The retina is the only organ projecting external light to the suprachiasmatic nucleus. Cholecystokinin receptor type A (Cckar/Cckar) is one of the essential factors for light reception in retinal cells. As there was a lack of literature on the matter, we aimed to elucidate the cause of the time-dependent phase change in clock gene expression. We found that Cckar mRNA expression in retinal cells exhibited diurnal variations. The rhythm of expression of the clock gene Per1/Per2 in retinal cells was altered in Cckar -/- mice. The light sensitivity of retinal cells was evaluated in wild-type mice, which showed c-Fos was activated in the ganglion cell layer more than in the inner granular layer. This increase in the number of c-Fos-positive cells was suppressed by lorglumide, a Cckar antagonist. Treatment of rat retina primary cells with lorglumide suppressed Per2 transcription, which was altered in a time-dependent manner relative to the Per2 expression. Light irradiation studies in Cckar -/- mice did not exhibit an increase in Period expression in the suprachiasmatic nucleus. These results indicate that Cckar is among the factors that regulate the cycle of clock genes on the retina. Cckar knockout attenuates the light responsiveness of suprachiasmatic nucleus and reduces the expression amplitude of Period genes in the retina. Thus, Cckar may contribute to entrainment of the light environment and maintenance of the expression cycle of Period gene, which is one of the core clock genes.

Epigenetic repression of de novo cysteine synthetases induces intra-cellular accumulation of cysteine in hepatocarcinoma by up-regulating the cystine uptake transporter xCT.

BACKGROUND: The metabolic reprogramming of amino acids is critical for cancer cell growth and survival. Notably, intracellular accumulation of cysteine is often observed in various cancers, suggesting its potential role in alleviating the oxidative stress associated with rapid proliferation. The liver is the primary organ for cysteine biosynthesis, but much remains unknown about the metabolic alterations of cysteine and their mechanisms in hepatocellular carcinoma cells. METHODS: RNA-seq data from patients with hepatocarcinoma were analyzed using the TNMplot database. The underlying mechanism of the oncogenic alteration of cysteine metabolism was studied in mice implanted with BNL 1ME A.7 R.1 hepatocarcinoma. RESULTS: Database analysis of patients with hepatocellular carcinoma revealed that the expression of enzymes involved in de novo cysteine synthesis was down-regulated accompanying with increased expression of the cystine uptake transporter xCT. Similar alterations in gene expression have also been observed in a syngeneic mouse model of hepatocarcinoma. The enhanced expression of DNA methyltransferase in murine hepatocarcinoma cells caused methylation of the upstream regions of cysteine synthesis genes, thereby repressing their expression. Conversely, suppression of de novo cysteine synthesis in healthy liver cells induced xCT expression by up-regulating the oxidative-stress response factor NRF2, indicating that reduced de novo cysteine synthesis repulsively increases cystine uptake via enhanced xCT expression, leading to intracellular cysteine accumulation. Furthermore, the pharmacological inhibition of xCT activity decreased intracellular cysteine levels and suppressed hepatocarcinoma tumor growth in mice. CONCLUSIONS: Our findings indicate an underlying mechanism of the oncogenic alteration of cysteine metabolism in hepatocarcinoma and highlight the efficacy of alteration of cysteine metabolism as a viable therapeutic target in cancer.

Macrophages modulate mesenchymal stem cell function via tumor necrosis factor alpha in tooth extraction model.

Mesenchymal stem cells (MSCs) and macrophages collaboratively contribute to bone regeneration after injury. However, detailed mechanisms underlying the interaction between MSCs and inflammatory macrophages (M1) remain unclear. A macrophage-depleted tooth extraction model was generated in 5-wk-old female C57BL/6J mice using clodronate liposome (12.5 mg/kg/mouse, intraperitoneally) or saline injection (control) before maxillary first molar extraction. Mice were sacrificed on days 1, 3, 5, 7, and 10 after tooth extraction (n = 4). Regenerated bone volume evaluation of tooth extraction socket (TES) and histochemical analysis of CD80+M1, CD206+M2 (anti-inflammatory macrophages), PDGFRα+MSC, and TNF-α+ cells were performed. In vitro, isolated MSCs with or without TNF-α stimulation (10 ng/mL, 24 h, n = 3) were bulk RNA-sequenced (RNA-Seq) to identify TNF-α stimulation-specific MSC transcriptomes. Day 7 micro-CT and HE staining revealed significantly lower mean bone volume (clodronate vs control: 0.01 mm3 vs 0.02 mm3, p<.0001) and mean percentage of regenerated bone area per total TES in clodronate group (41.97% vs 54.03%, p<.0001). Clodronate group showed significant reduction in mean number of CD80+, TNF-α+, PDGFRα+, and CD80+TNF-α+ cells on day 5 (306.5 vs 558.8, p<.0001; 280.5 vs 543.8, p<.0001; 365.0 vs 633.0, p<.0001, 29.0 vs 42.5, p<.0001), while these cells recovered significantly on day 7 (493.3 vs 396.0, p=.0004; 479.3 vs 384.5, p=.0008; 593.0 vs 473.0, p=.0010, 41.0 vs 32.5, p=.0003). RNA-Seq analysis showed that 15 genes (|log2FC| > 5.0, log2TPM > 5) after TNF-α stimulation were candidates for regulating MSC's immunomodulatory capacity. In vivo, Clec4e and Gbp6 are involved in inflammation and bone formation. Clec4e, Gbp6, and Cxcl10 knockdown increased osteogenic differentiation of MSCs in vitro. Temporal reduction followed by apparent recovery of TNF-α-producing M1 macrophages and MSCs after temporal macrophage depletion suggests that TNF-α activated MSCs during TES healing. In vitro mimicking the effect of TNF-α on MSCs indicated that there are 15 candidate MSC genes for regulation of immunomodulatory capacity.

Inhibition of dynamin-related protein 1-filamin interaction improves systemic glucose metabolism.

BACKGROUND AND PURPOSE: Maintaining mitochondrial quality is attracting attention as a new strategy to treat diabetes and diabetic complications. We previously reported that mitochondrial hyperfission by forming a protein complex between dynamin-related protein (Drp) 1 and filamin, mediates chronic heart failure and cilnidipine, initially developed as an L/N-type Ca2+ channel blocker, improves heart failure by inhibiting Drp1-filamin protein complex. We investigated whether cilnidipine improves hyperglycaemia of various diabetic mice models. EXPERIMENTAL APPROACH: Retrospective analysis focusing on haemoglobin A1c (HbA1c) was performed in hypertensive and hyperglycaemic patients taking cilnidipine and amlodipine. After developing diabetic mice by streptozotocin (STZ) treatment, an osmotic pump including drug was implanted intraperitoneally, followed by weekly measurements of blood glucose levels. Mitochondrial morphology was analysed by electron microscopy. A Ca2+ channel-insensitive cilnidipine derivative (1,4-dihydropyridine [DHP]) was synthesized and its pharmacological effect was evaluated using obese (ob/ob) mice fed with high-fat diet (HFD). KEY RESULTS: In patients, cilnidipine was superior to amlodipine in HbA1c lowering effect. Cilnidipine treatment improved systemic hyperglycaemia and mitochondrial morphological abnormalities in STZ-exposed mice, without lowering blood pressure. Cilnidipine failed to improve hyperglycaemia of ob/ob mice, with suppressing insulin secretion. 1,4-DHP improved hyperglycaemia and mitochondria abnormality in ob/ob mice fed HFD. 1,4-DHP and cilnidipine improved basal oxygen consumption rate of HepG2 cells cultured under 25 mM glucose. CONCLUSION AND IMPLICATIONS: Inhibition of Drp1-filamin protein complex formation becomes a new strategy for type 2 diabetes treatment.

Hericenone C attenuates the second phase of formalin-induced nociceptive behavior by suppressing the accumulation of CD11c-positive cells in the paw epidermis via phosphorylated P65.

Hericenone C is one of the most abundant secondary metabolites derived from Hericium erinaceus, under investigation for medicinal properties. Here, we report that Hericenone C inhibits the second phase of formalin-induced nociceptive behavior in mice. As the second phase is involved in inflammation, in a mechanistic analysis on cultured cells targeting NF-κB response element (NRE): luciferase (Luc)-expressing cells, lipopolysaccharide (LPS)-induced NRE::Luc luciferase activity was found to be significantly inhibited by Hericenone C. Phosphorylation of p65, which is involved in the inflammatory responses of the NF-κB signaling pathway, was also induced by LPS and significantly reduced by Hericenone C. Additionally, in mice, the number of CD11c-positive cells increased in the paw during the peak of the second phase of the formalin test, which decreased upon Hericenone C intake. Our findings confirm the possibility of Hericenone C as a novel therapeutic target for pain-associated inflammation.

Age-Related Effects on MSC Immunomodulation, Macrophage Polarization, Apoptosis, and Bone Regeneration Correlate with IL-38 Expression.

Mesenchymal stem cells (MSCs) are known to promote tissue regeneration and suppress excessive inflammation caused by infection or trauma. Reported evidence indicates that various factors influence the expression of MSCs' endogenous immunomodulatory properties. However, the detailed interactions of MSCs with macrophages, which are key cells involved in tissue repair, and their regulatory mechanisms are not completely understood. We herein investigated how age-related immunomodulatory impairment of MSCs alters the interaction of MSCs with macrophages during bone healing using young (5-week old) and aged (50-week old) mice. To clarify the relationship between inflammatory macrophages (M1) and MSCs, their spatiotemporal localization at the bone healing site was investigated by immunostaining, and possible regulatory mechanisms were analyzed in vitro co-cultures. Histomorphometric analysis revealed an accumulation of M1 and a decrease in MSC number at the healing site in aged mice, which showed a delayed bone healing. In in vitro co-cultures, MSCs induced M1 apoptosis through cell-to-cell contact but suppressed the gene expression of pro-inflammatory cytokines by soluble factors secreted in the culture supernatant. Interestingly, interleukin 38 (Il-38) expression was up-regulated in M1 after co-culture with MSCs. IL-38 suppressed the gene expression of inflammatory cytokines in M1 and promoted the expression of genes associated with M1 polarization to anti-inflammatory macrophages (M2). IL-38 also had an inhibitory effect on M1 apoptosis. These results suggest that MSCs may induce M1 apoptosis, suppress inflammatory cytokine production by M1, and induce their polarization toward M2. Nevertheless, in aged conditions, the decreased number and immunomodulatory function of MSCs could be associated with a delayed M1 clearance (i.e., apoptosis and/or polarization) and consequent delayed resolution of the inflammatory phase. Furthermore, M1-derived IL-38 may be associated with immunoregulation in the tissue regeneration site.

Circadian rhythms in CYP2A5 expression underlie the time-dependent effect of tegafur on breast cancer.

The chemotherapeutic agent tegafur, a prodrug that prolongs the half-life of fluorouracil (5-FU), exerts antitumor effects against various cancers. Since tegafur is metabolized to 5-FU by CYP2A6 in the liver, the expression of CYP2A6 determines the effect of tegafur. Here, we report that the expression rhythm of Cyp2a5, a homolog of human CYP2A6, in female mice causes dosing time-dependent differences in tegafur metabolism. In the livers of female mice, CYP2A5 expression showed a circadian rhythm, peaking during the dark period. This rhythm is regulated by RORA, a core clock component, and abrogation of the CYP2A5 activity abolished the time-dependent difference in the rate of tegafur metabolism in female mice. Furthermore, administration of tegafur to mice transplanted with 4T1 breast cancer cells during the dark period suppressed increases in tumor size compared to female mice treated during the light period. Our findings reveal a novel relationship between 5-FU prodrugs and circadian clock machinery, potentially influencing antitumor effects, and contributing to the development of time-aware chemotherapy regimens for breast cancer.

Hematopoietic Prostaglandin D Synthase Is Increased in Mast Cells and Pericytes in Autopsy Myocardial Specimens from Patients with Duchenne Muscular Dystrophy.

The leading cause of death for patients with Duchenne muscular dystrophy (DMD), a progressive muscle disease, is heart failure. Prostaglandin (PG) D2, a physiologically active fatty acid, is synthesized from the precursor PGH2 by hematopoietic prostaglandin D synthase (HPGDS). Using a DMD animal model (mdx mice), we previously found that HPGDS expression is increased not only in injured muscle but also in the heart. Moreover, HPGDS inhibitors can slow the progression of muscle injury and cardiomyopathy. However, the location of HPGDS in the heart is still unknown. Thus, this study investigated HPGDS expression in autopsy myocardial samples from DMD patients. We confirmed the presence of fibrosis, a characteristic phenotype of DMD, in the autopsy myocardial sections. Additionally, HPGDS was expressed in mast cells, pericytes, and myeloid cells of the myocardial specimens but not in the myocardium. Compared with the non-DMD group, the DMD group showed increased HPGDS expression in mast cells and pericytes. Our findings confirm the possibility of using HPGDS inhibitor therapy to suppress PGD2 production to treat skeletal muscle disorders and cardiomyopathy. It thus provides significant insights for developing therapeutic drugs for DMD.

Prostaglandin F2α Affects the Cycle of Clock Gene Expression and Mouse Behavior.

Prostaglandins are bioactive compounds, and the activation of their receptors affects the expression of clock genes. However, the prostaglandin F receptor (Ptgfr) has no known relationship with biological rhythms. Here, we first measured the locomotor period lengths of Ptgfr-KO (B6.129-Ptgfrtm1Sna) mice and found that they were longer under constant dark conditions (DD) than those of wild-type (C57BL/6J) mice. We then investigated the clock gene patterns within the suprachiasmatic nucleus in Ptgfr-KO mice under DD and observed a decrease in the expression of the clock gene cryptochrome 1 (Cry1), which is related to the circadian cycle. Moreover, the expression of Cry1, Cry2, and Period2 (Per2) mRNA were significantly altered in the mouse liver in Ptgfr-KO mice under DD. In the wild-type mouse, the plasma prostaglandin F2α (PGF2α) levels showed a circadian rhythm under a 12 h cycle of light-dark conditions. In addition, in vitro experiments showed that the addition of PTGFR agonists altered the amplitude of Per2::luc activity, and this alteration differed with the timing of the agonist addition. These results lead us to hypothesize that the plasma rhythm of PGF2α is important for driving clock genes, thus suggesting the involvement of PGF2α- and Ptgfr-targeting drugs in the biological clock cycle.

Inhibition of G protein-coupled receptor 68 using homoharringtonine attenuates chronic kidney disease-associated cardiac impairment.

Chronic kidney disease (CKD) induces cardiac inflammation and fibrosis and reduces survival. We previously demonstrated that G protein-coupled receptor 68 (GPR68) promotes cardiac inflammation and fibrosis in mice with 5/6 nephrectomy (5/6Nx) and patients with CKD. However, no method of GPR68 inhibition has been found that has potential for therapeutic application. Here, we report that Cephalotaxus harringtonia var. nana extract and homoharringtonine ameliorate cardiac inflammation and fibrosis under CKD by suppressing GPR68 function. Reagents that inhibit the function of GPR68 were explored by high-throughput screening using a medicinal plant extract library (8,008 species), and we identified an extract from Cephalotaxus harringtonia var. nana as a GPR68 inhibitor that suppresses inflammatory cytokine production in a GPR68 expression-dependent manner. Consumption of the extract inhibited inflammatory cytokine expression and cardiac fibrosis and improved the decreased survival attributable to 5/6Nx. Additionally, homoharringtonine, a cephalotaxane compound characteristic of C. harringtonia, inhibited inflammatory cytokine production. Homoharringtonine administration in drinking water alleviated cardiac fibrosis and improved heart failure and survival in 5/6Nx mice. A previously unknown effect of C. harringtonia extract and homoharringtonine was revealed in which GPR68-dependent inflammation and cardiac dysfunction were suppressed. Utilizing these compounds could represent a new strategy for treating GPR68-associated diseases, including CKD.

Suppression of neuropathic pain in the circadian clock-deficient Per2m/m mice involves up-regulation of endocannabinoid system.

Neuropathic pain often results from injuries and diseases that affect the somatosensory system. Disruption of the circadian clock has been implicated in the exacerbation of the neuropathic pain state. However, in this study, we report that mice deficient in a core clock component Period2 (Per2m/m mice) fail to develop tactile pain hypersensitivity even following peripheral nerve injury. Similar to male wild-type mice, partial sciatic nerve ligation (PSL)-Per2m/m male mice showed activation of glial cells in the dorsal horn of the spinal cord and increased expression of pain-related genes. Interestingly, α1D-adrenergic receptor (α1D-AR) expression was up-regulated in the spinal cord of Per2m/m mice, leading to increased production of 2-arachidonoylglycerol (2-AG), an endocannabinoid receptor ligand. This increase in 2-AG suppressed the PSL-induced tactile pain hypersensitivity. Furthermore, intraspinal dorsal horn injection of adeno-associated viral vectors expressing α1D-AR also attenuated pain hypersensitivity in PSL-wild-type male mice by increasing 2-AG production. Our findings reveal an uncovered role of the circadian clock in neuropathic pain disorders and suggest a link between α1D-AR signaling and the endocannabinoid system.

Time-Dependent Differences in Vancomycin Sensitivity of Macrophages Underlie Vancomycin-Induced Acute Kidney Injury.

Although vancomycin (VCM)-frequently used to treat drug-resistant bacterial infections-often induces acute kidney injury (AKI), discontinuation of the drug is the only effective treatment; therefore, analysis of effective avoidance methods is urgently needed. Here, we report the differences in the induction of AKI by VCM in 1/2-nephrectomized mice depending on the time of administration. Despite the lack of difference in the accumulation of VCM in the kidney between the light (ZT2) and dark (ZT14) phases, the expression of AKI markers due to VCM was observed only in the ZT2 treatment. Genomic analysis of the kidney suggested that the time of administration was involved in VCM-induced changes in monocyte and macrophage activity, and VCM had time-dependent effects on renal macrophage abundance, ATP activity, and interleukin (IL)-1ß expression. Furthermore, the depletion of macrophages with clodronate abolished the induction of IL-1ß and AKI marker expression by VCM administration at ZT2. This study provides evidence of the need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced AKI as well as the potential for macrophage-targeted AKI therapy. SIGNIFICANCE STATEMENT: There is a time of administration at which vancomycin (VCM)-induced renal injury is more and less likely to occur, and macrophages are involved in this difference. Therefore, there is a need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced acute kidney injury as well as the potential for macrophage-targeted acute kidney injury therapy.

Implications of biological clocks in pharmacology and pharmacokinetics of antitumor drugs.

Mammalians' circadian pacemaker resides in the paired suprachiasmatic nuclei (SCN). SCN control biological rhythms such as the sleep-wake rhythm and homeostatic functions of steroid hormones and their receptors. Alterations in these biological rhythms are implicated in the outcomes of pathogenic conditions such as depression, diabetes, and cancer. Chronotherapy is about optimizing treatment to combat risks and intensity of the disease symptoms that vary depending on the time of day. Thus, conditions/diseases such as allergic rhinitis, arthritis, asthma, myocardial infarction, congestive heart failure, stroke, and peptic ulcer disease, prone to manifest severe symptoms depending on the time of day, would be benefited from chronotherapy. Monitoring rhythm, overcoming rhythm disruption, and manipulating the rhythms from the viewpoints of underlying molecular clocks are essential to enhanced chronopharmacotherapy. New drugs focused on molecular clocks are being developed to improve therapeutics. In this review, we provide a critical summary of literature reports concerning (a) the rationale/mechanisms for time-dependent dosing differences in therapeutic outcomes and safety of antitumor drugs, (b) the molecular pathways underlying biological rhythms, and (c) the possibility of pharmacotherapy based on the intra- and inter-individual variabilities from the viewpoints of the clock genes.

Modulation of cell physiology by bispecific nanobodies enabling changes in the intracellular localization of organelle proteins.

Proteins localize to their respective organelles in cells. This localization is changed by activation or repression in response to signal transduction. Therefore, the appropriate intracellular localization of proteins is important for their functions to be exerted. However, difficulties are associated with controlling the localization of endogenous proteins. In the present study, we developed a conceptually new method of controlling the intracellular localization of endogenous proteins using bispecific nanobodies (BiNbs). BiNbs recognize proteins expressed in the inner membrane, cytoskeleton, nucleus, and peroxisomes, but not in mitochondria or endoplasmic reticulum. BiNbs designed to recognize ß-CATENIN and the intrinsic cytosolic protein VIMENTIN (3 × Flag ß-CAT-VIM BiNbs) decreased the ß-CATENIN-mediated transactivation of target genes by preventing its nuclear localization. Furthermore, 3 × Flag ß-CAT-VIM BiNbs suppressed the proliferation and invasion of the VIMENTIN-expressing breast cancer cell line MDA-MB-231, but not MDA-MB-468, in which the expression of VIMENTIN was defective. The present results revealed that changes in the intracellular localization of specific proteins by BiNbs modulated the physiology and functions of cells. The development of BiNbs to recognize proteins specifically expressed in target cells may be a useful approach for eliciting cell-selective effects.

Oral administration of vancomycin alleviates heart failure triggered by chronic kidney disease.

Chronic kidney disease (CKD) induces an imbalance in the intestinal microbiota, affecting various physiological functions and leading to cardiovascular inflammation and fibrosis. However, the cardiotoxic impact of intestinal microbiota-derived uremic substances in advanced renal dysfunction remains unexplored. Therefore, we developed a 5/6 nephrectomy (5/6Nx) mouse model to investigate the intestinal microbiota and the effects of administering vancomycin (VCM) on the microbiota and the cardiac pathology associated with CKD. Despite VCM administration after the development of irreversible glomerulosclerosis and tubulointerstitial fibrosis, blood indoxyl sulfate and phenyl sulfate levels, which are intestinal bacteria-derived uremic substances, brain natriuretic peptide levels, and the fibrotic area in the heart were decreased. Moreover, VCM administration prevented 5/6Nx-induced weight loss and prolonged survival time. Our findings suggest that VCM-induced changes in the intestinal microbiota composition ameliorate heart failure and improve survival rates by reducing intestinal microbiota-derived cardiotoxic substances despite advanced renal dysfunction. This highlights the potential of using the intestinal microbiota as a target to prevent and treat cardiovascular conditions associated with CKD.

Expanding the Chemistry of Dihaloacetamides as Tunable Electrophiles for Reversible Covalent Targeting of Cysteines.

The choice of an appropriate electrophile is crucial in the design of targeted covalent inhibitors (TCIs). In this report, we systematically investigated the glutathione (GSH) reactivity of various haloacetamides and the aqueous stability of their thiol adducts. Our findings revealed that dihaloacetamides cover a broad range of GSH reactivity depending on the combination of the halogen atoms and the structure of the amine scaffold. Among the dihaloacetamides, dichloroacetamide (DCA) exhibited slightly lower GSH reactivity than chlorofluoroacetamide (CFA). The DCA-thiol adduct is readily hydrolyzed under aqueous conditions, but it can stably exist in the solvent-sequestered binding pocket of the protein. These reactivity profiles of DCA were successfully exploited in the design of TCIs targeting noncatalytic cysteines of KRASG12C and EGFRL858R/T790M. These inhibitors exhibited strong antiproliferative activities against cancer cells. Our findings provide valuable insights for designing dihaloacetamide-based reversible covalent inhibitors.

Inhibition of Tumor-Derived C-C Motif Chemokine Ligand 2 Expression Attenuates Tactile Allodynia in NCTC 2472 Fibrosarcoma-Inoculated Mice.

Neuropathic pain associated with cancers is caused by tumor growth compressing and damaging nerves, which would also be enhanced by inflammatory factors through sensitizing nociceptor neurons. A troublesome hallmark symptom of neuropathic pain is hypersensitivity to innocuous stimuli, a condition known as "tactile allodynia", which is often refractory to NSAIDs and opioids. The involvement of chemokine CCL2 (monocyte chemoattractant protein-1) in cancer-evoked neuropathic pain is well established, but opinions remain divided as to whether CCL2 is involved in the production of tactile allodynia with tumor growth. In this study, we constructed Ccl2 knockout NCTC 2472 (Ccl2-KO NCTC) fibrosarcoma cells and conducted pain behavioral test using Ccl2-KO NCTC-implanted mice. Implantation of naïve NCTC cells around the sciatic nerves of mice produced tactile allodynia in the inoculated paw. Although the growth of Ccl2 KO NCTC-formed tumors was comparable to that of naïve NCTC-formed tumors, Ccl2-KO NCTC-bearing mice failed to show tactile pain hypersensitivity, suggesting the involvement of CCL2 in cancer-induced allodynia. Subcutaneous administration of controlled-release nanoparticles containing the CCL2 expression inhibitor NS-3-008 (1-benzyl-3-hexylguanidine) significantly attenuated tactile allodynia in naïve NCTC-bearing mice accompanied by a reduction of CCL2 content in tumor masses. Our present findings suggest that inhibition of CCL2 expression in cancer cells is a useful strategy to attenuate tactile allodynia induced by tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for the treatment of cancer-evoked neuropathic pain. SIGNIFICANCE STATEMENT: The blockade of chemokine/receptor signaling, particularly for C-C motif chemokine ligand 2 (CCL2) and its high-affinity receptor C-C chemokine receptor type 2 (CCR2), has been implicated to attenuate cancer-induced inflammatory and nociceptive pain. This study demonstrated that continuous inhibition of CCL2 production from cancer cells also prevents the development of tactile allodynia associated with tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for management of cancer-evoked tactile allodynia.

The scaffold protein PDZK1 governs diurnal localization of CNT2 on the plasma membrane in mouse intestinal epithelial cells.

Diurnal oscillations in the expression of several types of cell surface transporters have been demonstrated in the intestinal epithelial cells, which are mainly generated at transcriptional or degradation processes. Concentrative nucleoside transporter-2 (CNT2) is expressed at the apical site of intestinal epithelial cells and contributes to the uptake of nucleosides and their analogs from the intestinal lumen into the epithelial cells. In this study, we demonstrated that the localization of CNT2 protein in the plasma membrane of mouse intestinal epithelial cells exhibited a diurnal oscillation without changing its protein level in the whole cell. The scaffold protein PDZK1 interacted with CNT2 and stabilized its plasmalemmal localization. The expression of PDZK1 was under the control of molecular components of the circadian clock. Temporal accumulation of PDZK1 protein in intestinal epithelial cells enhanced the plasmalemmal localization of CNT2 at certain times of the day. The temporal increase in CNT2 protein levels at the plasma membrane also facilitated the uptake of adenosine into the intestinal epithelial cells. These results suggest a novel molecular mechanism for the diurnal localization of cell surface transporters and extend our understanding of the biological clock system that generates apparent physiological rhythms.

Fluorescence-Based Detection of Fatty Acid ß-Oxidation in Cells and Tissues Using Quinone Methide-Releasing Probes.

Detection of metabolic activity enables us to reveal the inherent metabolic state of cells and elucidate mechanisms underlying cellular homeostasis and growth. However, a fluorescence approach for the study of metabolic pathways is still largely unexplored. Herein, we have developed a new chemical probe for the fluorescence-based detection of fatty acid ß-oxidation (FAO), a key process in lipid catabolism, in cells and tissues. This probe serves as a substrate of FAO and forms a reactive quinone methide (QM) as a result of metabolic reactions. The liberated QM is covalently captured by intracellular proteins, and subsequent bio-orthogonal ligation with a fluorophore enables fluorescence analysis. This reaction-based sensing allowed us to detect FAO activity in cells at a desired emission wavelength using diverse analytical techniques including fluorescence imaging, in-gel fluorescence activity-based protein profiling (ABPP), and fluorescence-activated cell sorting (FACS). The probe was able to detect changes in FAO activity induced by chemical modulators in cultured cells. The probe was further employed for fluorescence imaging of FAO in mouse liver tissues and revealed the metabolic heterogeneity of FAO activity in hepatocytes by the combination of FACS and gene expression analysis, highlighting the utility of our probe as a chemical tool for fatty acid metabolism research.

Population Pharmacokinetic Analysis of Drug-Drug Interactions Between Perampanel and Carbamazepine Using Enzyme Induction Model in Epileptic Patients.

BACKGROUND: Perampanel (PER) is an oral antiepileptic drug and its concomitant use with carbamazepine (CBZ) leads to decreased PER concentrations. However, the magnitude of its influence may vary, depending on the dynamics of the enzyme induction properties of CBZ. This study aimed to develop a population pharmacokinetic (PPK) model considering the dynamics of enzyme induction and evaluate the effect of CBZ on PER pharmacokinetics. METHODS: We retrospectively collected data on patient background, laboratory tests, and prescribed drugs from electronic medical records. We developed 2 PPK models incorporating the effect of CBZ-mediated enzyme induction to describe time-concentration profiles of PER using the following different approaches: (1) treating the concomitant use of CBZ as a categorical covariate (empirical PPK model) and (2) incorporating the time-course of changes in the amount of enzyme by CBZ-mediated induction (semimechanistic PPK model). The bias and precision of the predictions were investigated by calculating the mean error, mean absolute error, and root mean squared error. RESULTS: A total of 133 PER concentrations from 64 patients were available for PPK modelling. PPK analyses showed that the co-administration of CBZ increased the clearance of PER. Goodness-of-fit plots indicated a favorable description of the observed data and low bias. The mean error, mean absolute error, and root mean square error values based on the semimechanistic model were smaller than those obtained using the empirical PPK model for predicting PER concentrations in patients with CBZ. CONCLUSIONS: We developed 2 PPK models to describe PER pharmacokinetics based on different approaches, using electronic medical record data. Our PPK models support the use of PER in clinical practice.