Tenapanor: A new treatment option for hyperphosphatemia in end stage kidney disease.

PURPOSE: This narrative review explores the currently published studies that have evaluated tenapanor for the treatment of hyperphosphatemia in end-stage kidney disease (ESKD) patients on hemodialysis. This medication's new phosphate lowering mechanism of action reduces intestinal phosphate absorption predominantly through reduction of passive paracellular phosphate flux by inhibition of the sodium/hydrogen exporter isoform 3 (NHE3). Tenapanor additionally prevents active transcellular phosphate absorption compensation by decreasing the expression of sodium phosphorus 2b transport protein (NaPi2b). METHODS: A comprehensive search of the literature was conducted using PubMed and ClinicalTrials.gov search engines. The search term "tenapanor hyperphosphatemia" was used for study retrieval. Results were limited to studies published in the English language and excluded review articles. Human, animal, and in vitro studies were included. No date range was specified. RESULTS: A total of 11 primary studies were identified and included in this review, the largest human study of which enrolled 236 patients. Each study is presented in table format along with measured end points. CONCLUSIONS: Tenapanor is the first drug in its class that lowers hyperphosphatemia in ESKD patients through a novel mechanism of action involving paracellular inactive transport. Although more studies are needed, early results indicate that tenapanor may have a place in managing hyperphosphatemia in ESKD patients both as monotherapy and as an adjunct to existing phosphate binder therapy.

Dihydroartemisinin improves hypercholesterolemia in ovariectomized mice via enhancing vectorial transport of cholesterol and bile acids from blood to bile.

The increase of concentrations of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in the serum of postmenopausal women is the important risk factor of the high morbidity of cardiovascular diseases of old women worldwide. To test the anti-hypercholesterolemia function of dihydroartemisinin (DHA) in postmenopausal women, ovariectomized (OVX) mice were generated, and DHA were administrated to OVX mice for 4 weeks. The blood and liver tissues were collected for biochemical and histological tests respectively. The mRNA and protein expression levels of genes related to metabolism and transport of cholesterol, bile acid and fatty acid in the liver or ileum were checked through qPCR and western blot. DHA could significantly reduce the high concentrations of TC and LDL-C in the serum and the lipid accumulation in the liver of ovariectomized mice. The expression of ABCG5/8 was reduced in liver of OVX mice, and DHA could up-regulate the expression of them. Genes of transport proteins for bile salt transport from blood to bile, including Slc10a1, Slco1b2 and Abcb11, were also significantly up-regulated by DHA. DHA also down-regulated the expression of Slc10a2 in the ileum of OVX mice to reduce the absorption of bile salts. Genes required for fatty acid synthesis and uptake, such as Fasn and CD36, were reduced in the liver of OVX mice, and DHA administration could significantly up-regulate the expression of them. These results demonstrated that DHA could improve hypercholesterolemia in OVX mice through enhancing the vectorial transport of cholesterol and bile acid from blood to bile.

Kinetic analysis of cystine uptake and inhibition pattern of sulfasalazine in A549 cells.

Cystine/glutamate transporter (xCT) is an antiporter involved in cystine uptake and glutamate efflux. However, there are very few reports regarding the kinetic analysis of xCT for cystine uptake using cancer cell lines, as well as the inhibition pattern of sulfasalazine, an inhibitor of xCT, for cystine uptake. Therefore, the purpose of this study was to clarify the kinetics of xCT in A549 cells, human lung cancer cells, and to reveal the inhibition pattern of sulfasalazine. Cystine uptake occurred in a time-dependent manner, with linear cystine uptake observed for 5 min. Additionally, sulfasalazine inhibited cystine uptake in a concentration-dependent manner, presenting an IC50 value of 24.7 ± 5.6 µM. Cystine uptake was saturated with increasing concentration, demonstrating Km and Vmax values of 179.4 ± 26.7 µM and 30.4 ± 2.3 nmol/min/mg protein, respectively. Moreover, during cystine uptake with sulfasalazine, Km and Vmax were >300 µM and 8.0 ± 1.5 nmol/min/mg protein, respectively, suggesting that sulfasalazine might demonstrate a mixed inhibition pattern. Furthermore, xCT siRNA decreased the xCT mRNA level and reduced cystine uptake. In conclusion, xCT was involved in the cystine uptake in A549 cells and sulfasalazine showed a mixed inhibition pattern to xCT.

A metabolic inhibitor arms macrophages to kill intracellular fungal pathogens by manipulating zinc homeostasis.

Macrophages deploy numerous strategies to combat invasion by microbes. One tactic is to restrict acquisition of diverse nutrients, including trace metals, a process termed nutritional immunity. Intracellular pathogens adapt to a resource-poor environment by marshaling mechanisms to harvest nutrients. Carbon acquisition is crucial for pathogen survival; compounds that reduce availability are a potential strategy to control intracellular replication. Treatment of macrophages with the glucose analog 2-deoxy-D-glucose (2-DG) armed phagocytes to eliminate the intracellular fungal pathogen Histoplasma capsulatum in vitro and in vivo. Killing did not rely on altering access to carbon-containing molecules or changes in ATP, ER stress, or autophagy. Unexpectedly, 2-DG undermined import of exogenous zinc into macrophages, decreasing the quantity of cytosolic and phagosomal zinc. The fungus perished as a result of zinc starvation. This change in metal ingress was not ascribed to a defect in a single importer; rather, there was a collective impairment in transporter activity. This effect promoted the antifungal machinery of macrophages and expanded the complexity of 2-DG activities far beyond manipulating glycolysis. Mechanistic metabolic studies employing 2-DG will have to consider its effect on zinc transport. Our preclinical data support consideration of this agent as a possible adjunctive therapy for histoplasmosis.

Structure-activity relationship and mechanism of flavonoids on the inhibitory activity of P-glycoprotein (P-gp)-mediated transport of rhodamine123 and daunorubicin in P-gp overexpressed human mouth epidermal carcinoma (KB/MDR) cells.

This study was aimed to investigate the inhibitory activity of flavonoids on P-glycoprotein (P-gp). Effects of 39 flavonoids on the cellular uptake (CU) of rhodamine123 (Rho) and daunomycin (DNR) were investigated in both parental KB and P-gp overexpressed KB/MDR cells. The inhibition mechanism of selected flavonoids was further investigated by measuring the ATPase activity and expression level of P-gp. Twelve flavonoids improved the uptake of Rho (↑RhoF) and nineteen flavonoids increased the uptake of DNR (↑DNRF) in KB/MDR cells with nine flavonoids overlapped. Structure-activity relationship (SAR) indicated that 8-OCH3, and 2'-OH have a negative effect on Rho and DNR transport. Whereas 5-OH, 5-OCH3, 6-OH, 7-OCH3, 3'-OH, and 4'-OH, are essential for inhibition of flavonoids on P-gp and reversing the resistance of Rho and DNR. Eleven selected flavonoids significantly induced the basal P-gp-ATPase activity but much lower than that induced by verapamil. Tangeretin, galangin, kaempferol, quercetin, and morin significantly reversed the ATPase activity stimulated by verapamil. Six of eleven flavonoids significantly decreased P-gp expression, whereas three flavonoids slightly increased P-gp expression. These results provide valuable information that flavonoids can effectively reverse multidrug resistance of P-gp-mediated transport of nutraceutical and drugs by co-administration.

Tetrahydrocannabinol and Its Major Metabolites Are Not (or Are Poor) Substrates or Inhibitors of Human P-Glycoprotein [ATP-Binding Cassette (ABC) B1] and Breast Cancer Resistance Protein (ABCG2).

(-)-Δ9-Tetrahydrocannabinol (THC) is the primary psychoactive constituent of cannabis. In humans, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) are psychoactive and nonpsychoactive circulating metabolites of THC, respectively. Whether these cannabinoids are substrates or inhibitors of human P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) is unknown. Previous animal studies suggest that THC and its metabolites could be substrates of these transporters. Therefore, we performed Transwell, cellular accumulation, and vesicular transport assays, at pharmacologically relevant concentrations of these cannabinoids, using Madin-Darby canine kidney (MDCK) II cells or plasma membrane vesicles overexpressing human P-gp or BCRP. Neither THC nor 11-OH-THC was found to be a substrate or inhibitor of P-gp or BCRP. The efflux ratio of THC-COOH in MDCKII-BCRP cells was 1.6, which was significantly decreased to 1.0 by the BCRP inhibitor Ko143. Likewise, cellular accumulation of THC-COOH was significantly increased 1.6-fold in the presence versus absence of Ko143. THC-COOH also significantly inhibited BCRP-mediated transport of Lucifer yellow, a BCRP substrate; however, THC-COOH was neither a substrate nor an inhibitor of P-gp. Collectively, these results indicate that THC and 11-OH-THC are not substrates or inhibitors (at pharmacologically relevant concentrations) of either P-gp or BCRP. THC-COOH is a weak substrate and inhibitor of BCRP, but not of P-gp. Accordingly, we predict that P-gp/BCRP will not modulate the disposition of these cannabinoids in humans. In addition, use of these cannabinoids will not result in P-gp- or BCRP-based drug interactions. SIGNIFICANCE STATEMENT: This study systematically investigated whether Δ9-tetrahydrocannabinol (THC) and its major metabolites, 11-hydroxy-THC and 11-nor-9-carboxy-THC, are substrates and/or inhibitors of human P-gp and BCRP at pharmacologically relevant concentrations. The results obtained are highly valuable for mechanistic understanding and prediction of the roles of P-gp and BCRP in determining the human pharmacokinetics, tissue distribution, and drug interactions of cannabinoids.

Physiologically Based Pharmacokinetic Modeling of Transporter-Mediated Hepatic Disposition of Imaging Biomarker Gadoxetate in Rats.

Physiologically based pharmacokinetic (PBPK) models are increasingly used in drug development to simulate changes in both systemic and tissue exposures that arise as a result of changes in enzyme and/or transporter activity. Verification of these model-based simulations of tissue exposure is challenging in the case of transporter-mediated drug-drug interactions (tDDI), in particular as these may lead to differential effects on substrate exposure in plasma and tissues/organs of interest. Gadoxetate, a promising magnetic resonance imaging (MRI) contrast agent, is a substrate of organic-anion-transporting polypeptide 1B1 (OATP1B1) and multidrug resistance-associated protein 2 (MRP2). In this study, we developed a gadoxetate PBPK model and explored the use of liver-imaging data to achieve and refine in vitro-in vivo extrapolation (IVIVE) of gadoxetate hepatic transporter kinetic data. In addition, PBPK modeling was used to investigate gadoxetate hepatic tDDI with rifampicin i.v. 10 mg/kg. In vivo dynamic contrast-enhanced (DCE) MRI data of gadoxetate in rat blood, spleen, and liver were used in this analysis. Gadoxetate in vitro uptake kinetic data were generated in plated rat hepatocytes. Mean (%CV) in vitro hepatocyte uptake unbound Michaelis-Menten constant (Km,u) of gadoxetate was 106 µM (17%) (n = 4 rats), and active saturable uptake accounted for 94% of total uptake into hepatocytes. PBPK-IVIVE of these data (bottom-up approach) captured reasonably systemic exposure, but underestimated the in vivo gadoxetate DCE-MRI profiles and elimination from the liver. Therefore, in vivo rat DCE-MRI liver data were subsequently used to refine gadoxetate transporter kinetic parameters in the PBPK model (top-down approach). Active uptake into the hepatocytes refined by the liver-imaging data was one order of magnitude higher than the one predicted by the IVIVE approach. Finally, the PBPK model was fitted to the gadoxetate DCE-MRI data (blood, spleen, and liver) obtained with and without coadministered rifampicin. Rifampicin was estimated to inhibit active uptake transport of gadoxetate into the liver by 96%. The current analysis highlighted the importance of gadoxetate liver data for PBPK model refinement, which was not feasible when using the blood data alone, as is common in PBPK modeling applications. The results of our study demonstrate the utility of organ-imaging data in evaluating and refining PBPK transporter IVIVE to support the subsequent model use for quantitative evaluation of hepatic tDDI.

Low-Dose Acrolein, an Endogenous and Exogenous Toxic Molecule, Inhibits Glucose Transport via an Inhibition of Akt-Regulated GLUT4 Signaling in Skeletal Muscle Cells.

Urinary acrolein adduct levels have been reported to be increased in both habitual smokers and type-2 diabetic patients. The impairment of glucose transport in skeletal muscles is a major factor responsible for glucose uptake reduction in type-2 diabetic patients. The effect of acrolein on glucose metabolism in skeletal muscle remains unclear. Here, we investigated whether acrolein affects muscular glucose metabolism in vitro and glucose tolerance in vivo. Exposure of mice to acrolein (2.5 and 5 mg/kg/day) for 4 weeks substantially increased fasting blood glucose and impaired glucose tolerance. The glucose transporter-4 (GLUT4) protein expression was significantly decreased in soleus muscles of acrolein-treated mice. The glucose uptake was significantly decreased in differentiated C2C12 myotubes treated with a non-cytotoxic dose of acrolein (1 µM) for 24 and 72 h. Acrolein (0.5-2 µM) also significantly decreased the GLUT4 expression in myotubes. Acrolein suppressed the phosphorylation of glucose metabolic signals IRS1, Akt, mTOR, p70S6K, and GSK3α/ß. Over-expression of constitutive activation of Akt reversed the inhibitory effects of acrolein on GLUT4 protein expression and glucose uptake in myotubes. These results suggest that acrolein at doses relevant to human exposure dysregulates glucose metabolism in skeletal muscle cells and impairs glucose tolerance in mice.

Drug-drug interaction between diclofenac and gamma-hydroxybutyric acid.

Gamma hydroxybutyric acid (GHB) has been approved clinically to treat excessive daytime sleepiness and cataplexy in patients with narcolepsy, alcohol and opioid withdrawal, and as an anesthetic. The use of GHB clinically is limited due to its high abuse potential. The absorption, clearance and tissue uptake of GHB is mediated by proton-dependent and sodium-coupled monocarboxylate transporters (MCTs and SMCTs) and inhibition of these transporters may result in a change in GHB pharmacokinetics and pharmacodynamics. Previous studies have reported that non-steroidal anti-inflammatory drugs (NSAIDs) may inhibit these monocarboxylate transporters. Therefore, the purpose of this work was to analyze the interaction between GHB (at a dose of 600 mg/kg i. v.) and the NSAID, diclofenac, by examining the effects of this drug on the in vivo pharmacokinetics and pharmacodynamics in rat studies. The pharmacodynamic effect evaluated was respiratory depression, a measure of toxicity observed by GHB at this dose. There was an improvement in the respiratory rate with diclofenac administration suggesting an effect of diclofenac on GHB toxicity. In vitro studies with rat blood brain endothelial cells (RBE4) that express MCT1 indicated that diclofenac can inhibit GHB transport with an IC50 of 10.6 µM at pH 7.4. In vivo studies found a decrease in brain GHB concentrations and a decrease in the brain-to-plasma concentration ratio following diclofenac treatment. With this study we can conclude that diclofenac and potentially other NSAIDs can inhibit the transport of GHB into the brain, therefore decreasing GHB's pharmacodynamic effects and toxicity.

Opportunities and Challenges in Tunneling Nanotubes Research: How Far from Clinical Application?

Tunneling nanotubes (TNTs) are recognized long membrane nanotubes connecting distance cells. In the last decade, growing evidence has shown that these subcellular structures mediate the specific transfer of cellular materials, pathogens, and electrical signals between cells. As intercellular bridges, they play a unique role in embryonic development, collective cell migration, injured cell recovery, cancer treatment resistance, and pathogen propagation. Although TNTs have been considered as potential drug targets for treatment, there is still a long way to go to translate the research findings into clinical practice. Herein, we emphasize the heterogeneous nature of TNTs by systemically summarizing the current knowledge on their morphology, structure, and biogenesis in different types of cells. Furthermore, we address the communication efficiency and biological outcomes of TNT-dependent transport related to diseases. Finally, we discuss the opportunities and challenges of TNTs as an exciting therapeutic approach by focusing on the development of efficient and safe drugs targeting TNTs.

Naphthylphthalamic acid associates with and inhibits PIN auxin transporters.

N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar) transport of the hormone auxin in plants. For decades, it has been a pivotal tool in elucidating the unique polar auxin transport-based processes underlying plant growth and development. Its exact mode of action has long been sought after and is still being debated, with prevailing mechanistic schemes describing only indirect connections between NPA and the main transporters responsible for directional transport, namely PIN auxin exporters. Here we present data supporting a model in which NPA associates with PINs in a more direct manner than hitherto postulated. We show that NPA inhibits PIN activity in a heterologous oocyte system and that expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to specific saturable NPA binding. We thus propose that PINs are a bona fide NPA target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent auxin transport and a logical parsimonious explanation for the known physiological effects of NPA on plant growth, as well as an alternative hypothesis to interpret past and future results. We also introduce PIN dimerization and describe an effect of NPA on this, suggesting that NPA binding could be exploited to gain insights into structural aspects of PINs related to their transport mechanism.

Effectiveness of Columbianadin, a Bioactive Coumarin Derivative, in Perturbing Transient and Persistent INa.

Columbianadin (CBN) is a bioactive coumarin-type compound with various biological activities. However, the action of CBN on the ionic mechanism remains largely uncertain, albeit it was reported to inhibit voltage-gated Ca2+ current or to modulate TRP-channel activity. In this study, whole-cell patch-clamp current recordings were undertaken to explore the modifications of CBN or other related compounds on ionic currents in excitable cells (e.g., pituitary GH3 cells and HL-1 atrial cardiomyocytes). GH3-cell exposure to CBN differentially decreased peak or late component of voltage-gated Na+ current (INa) with effective IC50 of 14.7 or 2.8 µM, respectively. The inactivation time course of INa activated by short depolarization became fastened in the presence of CBN with estimated KD value of 3.15 µM. The peak INa diminished by 10 µM CBN was further suppressed by subsequent addition of either sesamin (10 µM), ranolazine (10 µM), or tetrodotoxin (1 µM), but it was reversed by 10 µM tefluthrin (Tef); however, further application of 10 µM nimodipine failed to alter CBN-mediated inhibition of INa. CBN (10 µM) shifted the midpoint of inactivation curve of INa to the leftward direction. The CBN-mediated inhibition of peak INa exhibited tonic and use-dependent characteristics. Using triangular ramp pulse, the hysteresis of persistent INa enhanced by Tef was noticed, and the behavior was attenuated by subsequent addition of CBN. The delayed-rectifier or erg-mediated K+ current was mildly inhibited by 10 µM CBN, while it also slightly inhibited the amplitude of hyperpolarization-activated cation current. In HL-1 atrial cardiomyocytes, CBN inhibited peak INa and raised the inactivation rate of the current; moreover, further application of 10 µM Tef attenuated CBN-mediated decrease in INa. Collectively, this study provides an important yet unidentified finding revealing that CBN modifies INa in electrically excitable cells.

Targeting the Four Pillars of Enterohepatic Bile Salt Cycling; Lessons From Genetics and Pharmacology.

Bile salts play a pivotal role in lipid homeostasis, are sensed by specialized receptors, and have been implicated in various disorders affecting the gut or liver. They may play a role either as culprit or as potential panacea. Four very efficient transporters mediate most of the hepatic and intestinal bile salt uptake and efflux, and are each essential for the efficient enterohepatic circulation of bile salts. Starting from the intestinal lumen, conjugated bile salts cross the otherwise impermeable lipid bilayer of (primarily terminal ileal) enterocytes through the apical sodium-dependent bile acid transporter (gene SLC10A2) and leave the enterocyte through the basolateral heteromeric organic solute transporter, which consists of an alpha and beta subunit (encoded by SLC51A and SLC51B). The Na+ -taurocholate cotransporting polypeptide (gene SLC10A1) efficiently clears the portal circulation of bile salts, and the apical bile salt export pump (gene ABCB11) pumps the bile salts out of the hepatocyte into primary bile, against a very steep concentration gradient. Recently, individuals lacking either functional Na+ -taurocholate cotransporting polypeptide or organic solute transporter have been described, completing the quartet of bile acid transport deficiencies, as apical sodium-dependent bile acid transporter and bile salt export pump deficiencies were already known for years. Novel pathophysiological insights have been obtained from knockout mice lacking functional expression of these genes and from pharmacological transporter inhibition in mice or humans. Conclusion: We provide a concise overview of the four main bile salt transport pathways and of their status as possible targets of interventions in cholestatic or metabolic disorders.

Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication.

Salmonellosis and listeriosis together accounted for more than one third of foodborne illnesses in the United States and almost half the hospitalizations for gastrointestinal diseases in 2018 while tuberculosis afflicted over 10 million people worldwide causing almost 2 million deaths. Regardless of the intrinsic virulence differences among Listeria monocytogenes, Salmonella enterica and Mycobacterium tuberculosis, these intracellular pathogens share the ability to survive and persist inside the macrophage and other cells and thrive in iron rich environments. Interferon-gamma (IFN-γ) is a central cytokine in host defense against intracellular pathogens and has been shown to promote iron export in macrophages. We hypothesize that IFN-γ decreases iron availability to intracellular pathogens consequently limiting replication in these cells. In this study, we show that IFN-γ regulates the expression of iron-related proteins hepcidin, ferroportin, and ferritin to induce iron export from macrophages. Listeria monocytogenes, S. enterica, and M. tuberculosis infections significantly induce iron sequestration in human macrophages. In contrast, IFN-γ significantly reduces hepcidin secretion in S. enterica and M. tuberculosis infected macrophages. Similarly, IFN-γ-activated macrophages express higher ferroportin levels than untreated controls even after infection with L. monocytogenes bacilli; bacterial infection greatly down-regulates ferroportin expression. Collectively, IFN-γ significantly inhibits pathogen-associated intracellular iron sequestration in macrophages and consequently retards the growth of intracellular bacterial pathogens by decreasing iron availability.

Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB.

Resistance-nodulation-division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics.

Pyrroloquinoline Quinone Modifies Lipid Profile, but Not Insulin Sensitivity, of Palmitic Acid-Treated L6 Myotubes.

Pyrroloquinoline quinone (PQQ) is a novel stimulator of mitochondrial biogenesis and cellular energy metabolism. This is the first study investigating regulatory mechanisms and metabolic responses underlying PQQ's action in palmitate-exposed L6 myotubes. Particularly, we assessed alterations in lipid content and composition, expression of metabolic enzymes, and changes in glucose transport. The experiments were conducted using muscle cells subjected to short (2 h) and prolonged (24 h) incubation with PQQ in a sequence of pre- and post-palmitic acid (PA) exposure. We demonstrated the opposite effects of 2 and 24 h treatments with PQQ on lipid content, i.e., a decline in the level of free fatty acids and triacylglycerols in response to short-time PQQ incubation as compared to increases in diacylglycerol and triacylglycerol levels observed after 24 h. We did not demonstrate a significant impact of PQQ on fatty acid transport. The analysis of metabolic enzyme expression showed that the vast majority of PQQ-dependent alterations cumulated in the PA/PQQ 24 h group, including elevated protein amount of peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α), sirtuin-1 (SIRT1), phosphorylated 5'AMP-activated protein kinase (pAMPK), carnitine palmitoyltransferase I (CPT1), citrate synthase (CS), fatty acid synthase (FAS), and serine palmitoyltransferase, long chain base subunit 1 (SPT1). In conclusion, the results mentioned above indicate PQQ-dependent activation of both fatty acid oxidation and lipid synthesis in order to adapt cells to palmitic acid-rich medium, although PQQ did not attenuate insulin resistance in muscle cells.

Aesculetin Inhibits Osteoclastic Bone Resorption through Blocking Ruffled Border Formation and Lysosomal Trafficking.

For the optimal resorption of mineralized bone matrix, osteoclasts require the generation of the ruffled border and acidic resorption lacuna through lysosomal trafficking and exocytosis. Coumarin-type aesculetin is a naturally occurring compound with anti-inflammatory and antibacterial effects. However, the direct effects of aesculetin on osteoclastogenesis remain to be elucidated. This study found that aesculetin inhibited osteoclast activation and bone resorption through blocking formation and exocytosis of lysosomes. Raw 264.7 cells were differentiated in the presence of 50 ng/mL receptor activator of nuclear factor-κB ligand (RANKL) and treated with 1-10 µM aesculetin. Differentiation, bone resorption, and lysosome biogenesis of osteoclasts were determined by tartrate-resistance acid phosphatase (TRAP) staining, bone resorption assay, Western blotting, immunocytochemical analysis, and LysoTracker staining. Aesculetin inhibited RANKL-induced formation of multinucleated osteoclasts with a reduction of TRAP activity. Micromolar aesculetin deterred the actin ring formation through inhibition of induction of αvß3 integrin and Cdc42 but not cluster of differentiation 44 (CD44) in RANKL-exposed osteoclasts. Administering aesculetin to RANKL-exposed osteoclasts attenuated the induction of autophagy-related proteins, microtubule-associated protein light chain 3, and small GTPase Rab7, hampering the lysosomal trafficking onto ruffled border crucial for bone resorption. In addition, aesculetin curtailed cellular induction of Pleckstrin homology domain-containing protein family member 1 and lissencephaly-1 involved in lysosome positioning to microtubules involved in the lysosomal transport within mature osteoclasts. These results demonstrate that aesculetin retarded osteoclast differentiation and impaired lysosomal trafficking and exocytosis for the formation of the putative ruffled border. Therefore, aesculetin may be a potential osteoprotective agent targeting RANKL-induced osteoclastic born resorption for medicinal use.

SLC7A11 as a biomarker and therapeutic target in HPV-positive head and neck Squamous Cell Carcinoma.

Ferroptosis, a regulated form of cell necrosis was previously reported to be induced upon pharmacological targeting of the cystine transporter SLC7A11 in Head and neck Squamous Cell Carcinoma (HNSCC). Whether tumors arising in a context of chronic infection with Human Papillomavirus (HPV) are sensitive to ferroptosis is unknown. Using RNAseq data (both whole-tumor and single-cell sequencing) we report that HPV positive (HPV+ve) tumors have lower expression levels of SLC7A11 compared to HPV negative (HPV-ve) HNSCC. We examined in vitro the effect of erastin, a specific blocker of SLC7A11, applied on two HNSCC cell lines with stable expression of HPV16 E6 and E7 oncoproteins. We report a decrease in total GSH levels and an increased sensitivity to erastin-induced ferroptosis in E6-E7 cells. Cell sensitivity to ferroptosis was specificaly related to a defect in cystine transport since we found no difference in ferroptosis induced by the direct inhibition of GPX4, and N-Acetyl Cystein abolished the difference between WT and E6-E7-expressing cells. Our findings point to SLC7A11 as an HPV-related biomarker of potential therapeutic relevance in HNSCC. Targeting cystine import to promote ferroptosis might be a promising strategy against HPV+ve HNSCC. (188 words).

Electrogenesis in Plasma Membrane Fraction of Halotolerant Microalga Dunaliella maritima and Effects of N,N'-Dicyclohexylcarbodiimide.

The effects of N,N'-dicyclohexylcarbodiimide (DCCD), non-specific inhibitor of various transport systems functioning in biological membranes, on Na+-transporting P-type ATPase of the green halotolerant microalga Dunaliella maritima were studied in the experiments with vesicular plasma membranes isolated from the alga cells. The effects of DCCD on electrogenic/ion transport function of the enzyme and its ATP hydrolase activity were investigated. Electrogenic/ion transport function of the enzyme was recorded as a Na+-dependent generation of electric potential on the vesicle membranes with the help of the potential-sensitive probe oxonol VI. It was found that unlike many other ion-transporting ATPases, the Na+-ATPase of D. maritima is insensitive to DCCD. This agent did not inhibit either ATP hydrolysis catalyzed by this enzyme or its transport activity. At the same time DCCD affected the ability of the vesicle membranes to maintain electric potential generated by the D. maritima Na+-ATPase. The observed effects can be explained based on the assumption that DCCD interacts with the Na+/H+ antiporter in the plasma membrane of D. maritima.

Cadmium induces iron deficiency anemia through the suppression of iron transport in the duodenum.

Cadmium (Cd) is an environmental contaminant that triggers toxic effects in various tissues such as the kidney, liver, and lung. Cd can also cause abnormal iron metabolism, leading to anemia. Iron homeostasis is regulated by intestinal absorption. However, whether Cd affects the iron absorption pathway is unclear. We aimed to elucidate the relationship between the intestinal iron transporter system and Cd-induced iron deficiency anemia. C57BL/6J female and male mice, 129/Sv female mice, and DBA/2 female mice were given a single oral dose of CdCl2 by gavage. After 3 or 24 h, Cd decreased serum iron concentrations and inhibited the expression of iron transport-related genes in the duodenum. In particular, Cd decreased the levels of divalent metal transporter 1 and ferroportin 1 in the duodenum. In addition, human colon carcinoma Caco-2 cells were treated with CdCl2. After 72 h, Cd decreased the expression of iron transport-related factors in Caco-2 cells with a pattern similar to that seen in the murine duodenum. These findings suggest that Cd inhibits iron absorption through direct suppression of iron transport in duodenal enterocytes and contributes to abnormal iron metabolism.