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TMEM16A, a member of the Transmembrane protein 16 family, serves as the molecular basis for calcium activated chloride channels (CaCCs). We use RT-PCR to demonstrate the expression of TMEM16A in the neurons of Helicoverpa armigera, and record the CaCCs current of acute isolated neurons of H. armigera for the first time using patch clamp technology. In order to screen effective inhibitors of calcium-activated chloride channels, the inhibitory effects of four chloride channel inhibitors, CaCCinh-A01, NPPB, DIDS, and SITS, on CaCCs were compared. The inhibitory effects of the four inhibitors on the outward current of CaCCs were CaCCinh-A01 (10 μM, 56.31 %), NPPB (200 μM, 43.69 %), SITS (1 mM, 12.41 %) and DIDS (1 mM, 13.29 %). Among these inhibitors, CaCCinh-A01 demonstrated the highest efficacy as a blocker. To further explore whether calcium channel proteins can serve as potential targets of pyrethroids, we compared the effects of (type I) tefluthrin and (type II) deltamethrin on CaCCs. 10 μM and 100 μM tefluthrin can stimulate a large tail current in CaCCs, prolonging their deactivation time by 10.44 ms and 31.49 ms, and the V 0.5 shifted in the hyperpolarization by 2-8 mV. Then, deltamethrin had no obvious effect on the deactivation and activation of CaCCs. Therefore, CaCCs of H. armigera can be used as a potential target of pyrethroids, but type I and type II pyrethroids have different effects on CaCCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Drug resistance in infectious diseases developed by bacteria and fungi is an important issue since it is necessary to further develop novel compounds with biological activity that counteract this problem. In addition, new pharmaceutical compounds with lower secondary effects to treat cancer are needed. Coordination compounds appear to be accessible and promising alternatives aiming to overcome these problems. In this review, we summarize the recent literature on coordination compounds based on nitrobenzoic acid (NBA) as a ligand, its derivatives, and other nitro-containing ligands, which are widely employed owing to their versatility. Additionally, an analysis of crystallographic data is presented, unraveling the coordination preferences and the most effective crystallization methods to grow crystals of good quality. This underscores the significance of elucidating crystalline structures and utilizing computational calculations to deepen the comprehension of the electronic properties of coordination complexes., Competing Interests: The authors declare no conflict of interest.

We examined the mechanism by which 24( R )-ethyllophenol (MAB28) isolated from the branches of Morus alba caused neurite outgrowth in rat pheochromocytoma cells (PC12). MAB28 significantly promoted neurite outgrowth to a similar degree as the positive control, nerve growth factor (NGF). After incubation with MAB28 in PC12 cells, phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and cyclic AMP response element-binding protein was detected, but the time course of phosphorylation was different from that induced by NGF. The expression of chloride intracellular channel protein 3 (CLIC3) was significantly decreased by MAB28. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), an outward rectifying chloride channel inhibitor, significantly promoted neurite outgrowth in PC12 cells. These data suggested that MAB28 could induce neurite outgrowth by downregulating CLIC3 expression.

Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) delays and in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase (FAH) transgene in the porcine model of HT1. This therapy is well tolerated and provides stable long-term expression of FAH in pigs with HT1. Genomic integration displays a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach., (© 2022. The Author(s).)

Background: Apelin and its receptor angiotensin receptor - like 1 (APJ) are closely related to renal fibrosis, but their specific roles in renal fibrosis are still controversial. In this article, we discussed the role of Apelin/APJ system in renal fibrosis and its mechanism., Methods: Chronic intermittent hypoxia (CIH) rat model was established to induce the environment of renal fibrosis and a competitive antagonist of the APJ receptor ML221 was administered to CIH rats. The rats were divided into Control, CIH and ML221 groups. HE staining was used to detect the inflammatory injury and fibrosis of renal tissue. The expressions of renal fibrosis-related indicators transforming growth factor-β (TGF-β), α-smooth muscle actin (α-SMA) and Human type I collagen (Col-Ⅰ) were detected by immunohistochemistry. The levels of oxidative stress indexes reactive oxygen species (ROS), Malondialdehyde (MDA), Superoxide Dismutase (SOD) and inflammation-related indexes Interleukin (IL) -6, tumor necrosis factor-α (TNF-α) and IL-1β were detected by ELISA. At the same time, the levels of Apelin-13 and AngiotensinII (AngⅡ) were also measured by ELISA. Finally, western blot was used to detect the expression of Apelin pathway and renal fibrosis-related proteins. In addition, at the cellular level, we divided the cells into Control, CIH, Apelin-13 and Apelin-13+ML-221 groups to further verify the specific mechanisms at the cellular level., Results: The expression of Apeline-13 and its related pathways was significantly increased after the induction of CIH in rats. However, the degree of renal fibrosis in ML221 group was further significantly increased after inhibiting the expression of Apelin. At the cellular level, CIH model cells treated with Apelin-13 significantly reduced cell proliferation, oxidative stress and inflammatory response, and decreased the expression of fibrosis-related proteins, which can be reversed by ML221 administration., Conclusion: The increased reactivity of Apelin may be one of the protective mechanisms against renal fibrosis induced by CIH., (Copyright © 2021. Published by Elsevier Inc.)

Type 1 diabetes mellitus (T1DM) is a metabolic disorder with severe hyperglycemia, one of the complications of which is testicular dysfunctions, androgen deficiency and decreased male fertility. In the diabetic testes, the expression and signaling pathways of leptin and a number of other adipokines are significantly changed. However, there is no information on the localization and expression of adipokine, apelin and its receptor (APJ) in the diabetic testes, although there is information on the involvement of apelin in the regulation of reproductive functions. The aim of this study was to investigate the expression and localization of apelin and APJ in the testes of mice with streptozotocin-induced T1DM and to estimate the effects of agonist (apelin-13) and antagonist (ML221) of APJ on the testosterone production by diabetic testis explants in the in vitro conditions. We first detected the expression of apelin and its receptor in the mouse testes, and showed an increased intratesticular expression of apelin and APJ along with the reduced testosterone secretion in T1DM. Using imunohistochemical approach, we showed that apelin and APJ are localized in the Leydig and germ cells, and in diabetes, the amount of these proteins was significantly higher than in the control mice. The diabetic testes had a decrease in germ cell proliferation (the reduced PCNA and GCNA levels) and an increase in apoptosis (the increased active caspase-3 and decreased BCL2 levels). These results suggest an involvement of apelin and APJ in T1DM-induced testicular pathogenesis. Treatment of the cultured testis explants with ML221 significantly increased the testosterone secretion, whereas apelin-13 was ineffective. Thus, hyperapelinemia in the testes can significantly contribute to testicular pathogenesis in T1DM, and pharmacological inhibition of apelin receptors can improve testicular steroidogenesis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background and Aims: Apelin (APLN) is the endogenous ligand of its G protein-coupled receptor, apelin receptor (APJ). APLN serum levels are increased in human liver diseases. We evaluated whether the APLN-APJ axis regulates ductular reaction and liver fibrosis during cholestasis., Approach and Results: We measured the expression of APLN and APJ and serum APLN levels in human primary sclerosing cholangitis (PSC) samples. Following bile duct ligation (BDL) or sham surgery, male wild-type (WT) mice were treated with ML221 (APJ antagonist) or saline for 1 week. WT and APLN -/- mice underwent BDL or sham surgery for 1 week. Multidrug resistance gene 2 knockout (Mdr2 -/- ) mice were treated with ML221 for 1 week. APLN levels were measured in serum and cholangiocyte supernatants, and cholangiocyte proliferation/senescence and liver inflammation, fibrosis, and angiogenesis were measured in liver tissues. The regulatory mechanisms of APLN-APJ in (1) biliary damage and liver fibrosis were examined in human intrahepatic biliary epithelial cells (HIBEpiCs) treated with APLN and (2) hepatic stellate cell (HSC) activation in APLN-treated human HSC lines (HHSteCs). APLN serum levels and biliary expression of APLN and APJ increased in PSC samples. APLN levels were higher in serum and cholangiocyte supernatants from BDL and Mdr2 -/- mice. ML221 treatment or APLN -/- reduced BDL-induced and Mdr2 -/- -induced cholangiocyte proliferation/senescence, liver inflammation, fibrosis, and angiogenesis. In vitro, APLN induced HIBEpiC proliferation, increased nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, reactive oxygen species (ROS) generation, and extracellular signal-regulated kinase (ERK) phosphorylation. Pretreatment of HIBEpiCs with ML221, diphenyleneiodonium chloride (Nox4 inhibitor), N-acetyl-cysteine (NAC, ROS inhibitor), or PD98059 (ERK inhibitor) reduced APLN-induced cholangiocyte proliferation. Activation of HHSteCs was induced by APLN but reduced by NAC., Conclusions: The APLN-APJ axis induces cholangiocyte proliferation through Nox4/ROS/ERK-dependent signaling and HSC activation through intracellular ROS. Modulation of the APLN-APJ axis may be important for managing cholangiopathies., (© 2020 by the American Association for the Study of Liver Diseases.)

Oral squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a high incidence of recurrence and distant metastasis. However, the mechanism of epithelial to mesenchymal transition (EMT) during tumor progression and metastasis in OSCC has not yet been fully elucidated. It is well known that the Cl - channel controls cell volume and activates several signaling pathways for cell differentiation. The aim of the present study was to investigate the role of the Cl - channel on EMT in the OSC 20 cell line, which is an OSCC line. OSC-20 cells were cultured with low serum medium containing a Cl - channel blocker NPPB. Morphological changes, gene expression, immunoreactivity, cell volume, and signaling pathway of the NPPB-treated OSC-20 cells were evaluated. The NPPB-treated OSC-20 cells showed typical morphology of mesenchymal cells. The expression levels of the epithelial marker E-cadherin in the NPPB-treated OSC-20 cells were lower than those of the untreated and TGF-β1-treated OSC-20 cells. On the other hand, mesenchymal markers such as vimentin, ZEB1, and Snail, in the NPPB-treated OSC-20 cells were higher than those in the untreated and TGF-β1-treated OSC-20 cells. Furthermore, a large number of vimentin-positive cells also appeared in the NPPB-treated OSC-20 cells. Additionally, the cell volume of these cells was significantly increased compared to that of the untreated and TGF-β1-treated cells. Interestingly, NPPB did not activate the TGF-β/smad signaling pathway, but activated the Wnt/β-catenin signaling pathway. These results suggest that Cl - channel dysfunction promoted EMT via activation of the Wnt/β-catenin signaling pathway in OSCC., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cataracts have a high incidence and prevalence rate worldwide, and they are the leading cause of blindness. Lens epithelial cell (LEC) apoptosis is often analysed in cataract research since it is the pathological basis of cataracts, except for congenital cataract. Chloride channels are present in ocular tissues, such as in trabecular cells, LECs and other cells. They serve an important role in apoptosis and participate in endoplasmic reticulum (ER) stress and oxidative stress. However, their role in the apoptosis of LECs has not been discussed. The present study examined the effects of the chloride channel blocker 5‑nitro‑2‑(3‑phenylpropylamino) benzoic acid (NPPB) in human LECs (HLECs) to elucidate the role of NPPB in HLECs and investigate the role and mechanism of chloride channels in cataract formation. HLECs were exposed to NPPB. Cell survival rate was evaluated using Cell Counting Kit‑8 assays. Oxidative stress was detected as reactive oxygen species (ROS) in cells by using a ROS assay kit. Apoptosis was examined by assessing mitochondrial membrane potential and using a JC‑1 assay kit, and western blot analysis was performed to measure the expression levels of mitochondrial‑dependent apoptosis pathway‑associated proteins. ER stress was evaluated by determining the intracellular calcium ion fluorescence intensity, and western blot analysis was performed to measure ER stress‑associated protein expression. The results revealed that NPPB treatment decreased the viability of HLECs and increased apoptosis. Additionally, NPPB increased intracellular ROS levels, as well as the number of JC‑1 monomers and the protein expression levels of B‑cell lymphoma‑2 (Bcl‑2)‑associated X and cleaved caspase‑3, and decreased Bcl‑2 protein expression. NPPB increased intracellular calcium ions, the protein expression levels of activating transcription factor 6, JNK, C/EBP homologous protein and caspase‑12, and the phosphorylation of protein kinase R‑like endoplasmic reticulum kinase. N‑acetylcysteine and 4‑phenylbutyric acid inhibited NPPB‑induced oxidative stress, ER stress and apoptosis. Therefore, NPPB treatment decreased cell viability and promoted apoptosis of HLECs via the promotion of oxidative and ER stress.

Coenzyme Q 10 (CoQ, ubiquinone) is a redox-active lipid endogenously synthesized by the cells. The final stage of CoQ biosynthesis is performed at the mitochondrial level by the 'complex Q', where coq2 is responsible for the prenylation of the benzoquinone ring of the molecule. We report that the competitive coq2 inhibitor 4-nitrobenzoate (4-NB) decreased the cellular CoQ content and caused severe impairment of mitochondrial function in the T67 human glioma cell line. In parallel with the reduction in CoQ biosynthesis, the cholesterol level increased, leading to significant perturbation of the plasma membrane physicochemical properties. We show that 4-NB treatment did not significantly affect the cell viability, because of an adaptive metabolic rewiring toward glycolysis. Hypoxia-inducible factor 1α (HIF-1α) stabilization was detected in 4-NB-treated cells, possibly due to the contribution of both reduction in intracellular oxygen tension and ROS overproduction. Exogenous CoQ supplementation partially recovered cholesterol content, HIF-1α degradation, and ROS production, whereas only weakly improved the bioenergetic impairment induced by the CoQ depletion. Our data provide new insights on the effect of CoQ depletion and contribute to shed light on the pathogenic mechanisms of ubiquinone deficiency syndrome., (© 2020 Federation of European Biochemical Societies.)

Proteases are industrially important catalysts. They belong to a complex family of enzymes that perform highly focused proteolysis functions. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. In the present study, a novel extracellular neutral protease produced from Arthrospira platensis was detected and characterized. Its proteolytic activity was strongly activated by β-mercaptoethanol, 5,5-dithio-bis-(2-nitrobenzoic acid) and highly inhibited by Hg 2+ and Zn 2+ metal ions which support the fact that the studied protease belongs to the cysteine protease family. Using statistical modelling methodology, the logistic model has been selected to predict A. platensis growth-kinetic values. The optimal culture conditions for neutral protease production were found using Box-Behnken Design. The maximum experimental protease activities (159.79 U/mL) was achieved after 13 days of culture in an optimized Zarrouk medium containing 0.625 g/L NaCl, 0.625 g/L K 2 HPO 4 and set on 9.5 initial pH. The extracellular protease of A. platensis can easily be used in the food industry for its important activity at neutral pH and its low production cost since it is a valuation of the residual culture medium after biomass recovery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

For over two decades, nitisinone (NTBC) has been successfully used to manipulate the tyrosine degradation pathway and save the lives of many children with hereditary tyrosinaemia type 1. More recently, NTBC has been used to halt homogentisic acid accumulation in alkaptonuria (AKU) with evidence suggesting its efficacy as a disease modifying agent. NTBC-induced hypertyrosinaemia has been associated with cognitive impairment and potentially sight-threatening keratopathy. In the context of a non-lethal condition (ie, AKU), these serious risks call for an evaluation of the wider impact of NTBC on the tyrosine pathway. We hypothesised that NTBC increases the tyrosine pool size and concentrations in tissues. In AKU mice tyrosine concentrations of tissue homogenates were measured before and after treatment with NTBC. In humans, pulse injection with l-[ 13 C 9 ]tyrosine and l-[d 8 ]phenylalanine was used along with compartmental modelling to estimate the size of tyrosine pools before and after treatment with NTBC. We found that NTBC increased tyrosine concentrations in murine tissues by five to nine folds. It also significantly increased the tyrosine pool size in humans (P < .001), suggesting that NTBC increases tyrosine not just in serum but also in tissues (ie, acquired tyrosinosis). This study provides, for the first time, the experimental proof for the magnitude of NTBC-related acquired tyrosinosis which should be overcome to ensure the safe use of NTBC in AKU., (© 2020 SSIEM.)

Skin pigmentation is a highly heterogeneous trait with diverse consequences worldwide. SLC24A5, encoding a potent K + -dependent Na + /Ca 2+ exchanger, is among the known color-coding genes that participate in melanogenesis by maintaining pH in melanosomes. Deficient SLC24A5 activity results in oculocutaneous albinism (OCA) type 6 in humans. In this study, by utilizing a exome sequencing (ES) approach, we identified two new variants [p. (Gly110Arg) and p. (IIe189Ilefs*1)] of SLC24A5 cosegregating with the OCA phenotype, including nystagmus, strabismus, foveal hypoplasia, albinotic fundus, and vision impairment, in three large consanguineous Pakistani families. Both of these variants failed to rescue the pigmentation in zebrafish slc24a5 morphants, confirming the pathogenic effects of the variants. We also phenotypically characterized a commercially available zebrafish mutant line (slc24a5 ko ) that harbors a nonsense (p.Tyr208*) allele of slc24a5. Similar to morphants, homozygous slc24a5 ko mutants had significantly reduced melanin content and pigmentation. Next, we used these slc24a5 ko zebrafish mutants to test the efficacy of nitisinone, a compound known to increase ocular and fur pigmentation in OCA1 (TYR) mutant mice. Treatment of slc24a5 ko mutant zebrafish embryos with varying doses of nitisinone did not improve melanin production and pigmentation, suggesting that treatment with nitisinone is unlikely to be therapeutic in OCA6 patients., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In this work, 12 novel herbicidal ionic liquids (HILs) based on acifluorfen were prepared by pairing with the fluorescent hydrazides or different alkyl chains for increasing activities and reducing negative impacts on the aquatic environment. The results showed that the fluorescence of coumarin hydrazide in the HILs was applied as the internal and supplementary light source to meet the requirement of light wavelength range of acifluorfen, which improved the phytotoxicity of acifluorfen to weeds by enhancing singlet oxygen generation with increased sunlight utilization. The herbicidal activities of HILs were related positively with the length of chain of cation under high light intensity and depended mainly on the fluorescence characteristic of the cation under low light intensity, and the double salt IL forms of acifluorfen containing coumarin hydrazide and n -hexadecyltrimethylammonium had enhanced efficacies against broadleaf weeds in the field. Compared with acifluorfen sodium, HILs had lower water solubility, better surface activity, weaker mobility in soils, and higher decomposition temperature. These results demonstrated that HILs containing different cations provided a wider scope for fine-tuning of the physicochemical and biological properties of herbicides and established a promising way for the development of environmentally friendly herbicidal formulations.

5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a non-specific chloride channel blocker. Peritoneal adhesion is an inevitable complication of abdominal surgery and remains an important clinical problem, leading to chronic pain, intestinal obstruction, and female infertility. The aim of this study is to observe the effects of NPPB on peritoneal adhesions and uncover the underlying mechanism. The formation of postoperative peritoneal adhesions was induced by mechanical injury to the peritoneum of rats. MTT assay and wound-healing assay were used to evaluate proliferation and migration of primary cultured adhesion fibroblasts (AFB) respectively. Whole-cell chloride currents were measured using a fully automated patch-clamp workstation. Cell volume changes were monitored by light microscopy and video imaging. Our results demonstrated that NPPB could significantly prevent the formation of peritoneal adhesion in rats and inhibit the proliferation of AFB in a concentration-dependent manner. NPPB also reduced the migration of AFB cells with an IC 50 of 53.09 μM. A 47% hypotonic solution successfully activated the I Cl,vol in AFB cells. The current could be blocked by extracellular treatment with NPPB. Moreover, 100 μM NPPB almost completely eliminated the capacity of regulatory volume decrease (RVD) in these cells. These data indicate that NPPB could prevent the formation of postoperative peritoneal adhesions. The possible mechanism may be through the inhibition of the proliferation and migration of AFB cells by modulating I Cl,vol and cell volume. These results suggest a potential clinical use of NPPB for preventing the formation of peritoneal adhesions.

Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase deficiency that leads to homogentisic acid (HGA) accumulation, ochronosis and severe osteoarthropathy. Recently, nitisinone treatment, which blocks HGA formation, has been effective in AKU patients. However, a consequence of nitisinone is elevated tyrosine that can cause keratopathy. The effect of tyrosine and phenylalanine dietary restriction was investigated in nitisinone-treated AKU mice, and in an observational study of dietary intervention in AKU patients. Nitisinone-treated AKU mice were fed tyrosine/phenylalanine-free and phenylalanine-free diets with phenylalanine supplementation in drinking water. Tyrosine metabolites were measured pre-nitisinone, post-nitisinone, and after dietary restriction. Subsequently an observational study was undertaken in 10 patients attending the National Alkaptonuria Centre (NAC), with tyrosine >700 μmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine-free amino acid supplements. Elevated tyrosine (813 μmol/L) was significantly reduced in nitisinone-treated AKU mice fed a tyrosine/phenylalanine-free diet in a dose responsive manner. At 3 days of restriction, tyrosine was 389.3, 274.8, and 144.3 μmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine-free diet; at 3 days tyrosine was 757.3, 530.2, and 656.2 μmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (P = .002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine-free amino acid supplementation; 4 out of 10 patients achieved tyrosine <700 μmol/L. Tyrosine/phenylalanine dietary restriction significantly reduced nitisinone-induced tyrosinemia in mice, with phenylalanine restriction alone proving ineffective. Similarly, protein restriction significantly reduced circulating tyrosine in AKU patients., (© 2019 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Tyrosinemia type 1 (TT1) is a rare metabolic disease caused by a defect in the tyrosine degradation pathway. Neurocognitive deficiencies have been described in TT1 patients, that have, among others, been related to changes in plasma large neutral amino acids (LNAA) that could result in changes in brain LNAA and neurotransmitter concentrations. Therefore, this project aimed to investigate plasma and brain LNAA, brain neurotransmitter concentrations and behavior in C57 Bl/6 fumarylacetoacetate hydrolase deficient (FAH-/-) mice treated with 2-(2-nitro-4-trifluoromethylbenoyl)-1,3-cyclohexanedione (NTBC) and/or diet and wild-type mice. Plasma and brain tyrosine concentrations were clearly increased in all NTBC treated animals, even with diet ( p < 0.001). Plasma and brain phenylalanine concentrations tended to be lower in all FAH-/- mice. Other brain LNAA, were often slightly lower in NTBC treated FAH-/- mice. Brain neurotransmitter concentrations were usually within a normal range, although serotonin was negatively correlated with brain tyrosine concentrations ( p < 0.001). No clear behavioral differences between the different groups of mice could be found. To conclude, this is the first study measuring plasma and brain biochemistry in FAH-/- mice. Clear changes in plasma and brain LNAA have been shown. Further research should be done to relate the biochemical changes to neurocognitive impairments in TT1 patients.

We examined the molecular regulation of porphyrin biosynthesis and protective responses in transgenic rice (Oryza sativa) expressing Bradyrhizobium japonicum Fe-chelatase (BjFeCh) after treatment with acifluorfen (AF). During the photodynamic stress imposed by AF, transcript levels of BjFeCh in transgenic plants increased greatly; moreover, transcript levels of OsFeCh2 remained almost constant, whereas in wild type (WT) plants they were considerably down-regulated. In the heme branch, transgenic plants exhibited greater levels of OsFC and HO transcripts than WT plants in the untreated stems as well as in the AF-treated leaves and stems. Both WT and transgenic plants treated with AF substantially decreased transcript levels for all the genes in the chlorophyll branch, with less decline in transgenic plants. After AF treatment, ascorbate (Asc) content and the redox Asc state greatly decreased in leaves of WT plants; however, in transgenic plants both parameters remained constant in leaves and the Asc redox state increased by 20% in stems. In response to AF, the leaves of WT plants greatly up-regulated CatA, CatB, and GST compared to those of transgenic plants, whereas, in the stems, transgenic plants showed higher levels of CatA, CatC, APXb, BCH, and VDE. Photochemical quenching, q P , was considerably dropped by 31% and 18% in WT and transgenic plants, respectively in response to AF, whereas non-radiative energy dissipation through non-photochemical quenching increased by 77% and 38% in WT and transgenic plants, respectively. Transgenic plants treated with AF exhibited higher transcript levels of nucleus-encoded photosynthetic genes, Lhcb1 and Lhcb6, as well as levels of Lhcb6 protein compared to those of WT plants. Our study demonstrates that expression of BjFeCh in transgenic plants influences not only the regulation of porphyrin biosynthesis through maintaining higher levels of gene expression in the heme branch, but also the Asc redox function during photodynamic stress caused by AF., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The ciliary transport is controlled by two parameters of the ciliary beating, frequency (CBF) and amplitude. In this study, we developed a novel method to measure both CBF and ciliary bend distance (CBD, an index of ciliary beating amplitude) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which are prepared from patients contracting allergic rhinitis and chronic sinusitis. An application of Cl - -free NO 3 - solution or bumetanide (an inhibitor of Na + /K + /2Cl - cotransport), which decreases intracellular Cl - concentration ([Cl - ] i ), increased CBD, not CBF, at 37 °C; however, it increased both CBD and CBF at 25 °C. Conversely, addition of Cl - channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4-[[4-Oxo-2-thioxo-3-[3-trifluoromethyl]phenyl]-5-thiazolidinylidene]methyl] benzoic acid (CFTR(inh)-172)), which increase [Cl - ] i , decreased both CBD and CBF, suggesting that CFTR plays a crucial role for maintaining [Cl - ] i in these cells. We speculate that Cl - modulates activities of the molecular motors regulating both CBD and CBF in cHNECs. Moreover, application of the CO 2 /HCO 3 - -free solution did not change intracellular pH (pH i ), and addition of an inhibitor of carbonic anhydrase (acetazolamide) sustained pH i increase induced by the NH 4 + pulse, which transiently increased pH i in the absence of acetazolamide. These results indicate that the cHNEC produces a large amount of CO 2 , which maintains a constant pH i even under the CO 2 /HCO 3 - -free condition.

Systematic identification and quantification of active radical sites in a small molecule, pyrazolium 3,5-dinitrobenzoate:3,5-dinitrobenzoic acid as well as in the stable free radical (DPPH • ) were carried out by Fukui functions calculation using DFT functional with B3LYP/6-311++G(d,p) level of basis set. Bioactive Lewis acid-base compound, pyrazolium 3,5-dinitrobenzoate:3,5-dinitrobenzoic acid (PDNB:DNBA) has been synthesized and crystallized by slow evaporation - solution method at 30 °C. Various functional groups and the structural arrangements were ascertained from spectral and XRD analyses, respectively. UV-vis spectral analysis was used to find out the stability of the anticipated drug for about 60 min using methanol as a solvent. Stabilization of the compound was linked to the presence of enormous N-H…O, O-H…O and C-H…O hydrogen bonding interactions identified through Hirshfeld surface analysis. Chemical stability and reactivity of the drug were validated from theoretical optimization and HOMO-LUMO analysis. Active nucleophilic, electrophilic and radical sites of PDNB:DNBA were also identified from molecular electrostatic potential analysis. Inhibition of growth of pathogens in screening experiments by the proposed drug attests its suitability in biological applications. Antioxidant activity of the compound, PDNB:DNBA, endorses its aptness for scavenging reactive radicals. Fluorimetry experiments confirm hyperchromism in DNA binding analysis proving groove mode of binding. Molecular docking explored the various modes of intermolecular interactions of the drug with microbes as well as DNA., (Copyright © 2019. Published by Elsevier B.V.)

High mortality rates in ovarian cancer are due to late-stage diagnosis when extensive metastases are present, coupled with the eventual development of resistance to standard chemotherapy. There is, thus, an urgent need to identify targetable pathways to curtail this deadly disease. In this study, we show that the apelin receptor, APJ, is a viable target that promotes tumor progression of high-grade serous ovarian cancer (HGSOC). APJ is specifically overexpressed in tumor tissue, and is elevated in metastatic tissues compared with primary tumors. Importantly, increased APJ expression significantly correlates with decreased median overall survival (OS) by 14.7 months in patients with HGSOC. Using various ovarian cancer model systems, we demonstrate that APJ expression in cancer cells is both necessary and sufficient to increase prometastatic phenotypes in vitro , including proliferation, cell adhesion to various molecules of the extracellular matrix (ECM), anoikis resistance, migration, and invasion; and these phenotypes are efficiently inhibited by the APJ inhibitor, ML221. Overexpression of APJ also increases metastasis of ovarian cancer cells in vivo . Mechanistically, the prometastatic STAT3 pathway is activated downstream of APJ, and in addition to the ERK and AKT pathways, contributes to its aggressive phenotypes. Our findings suggest that the APJ pathway is a novel and viable target, with potential to curb ovarian cancer progression and metastasis. IMPLICATIONS: The APJ pathway is a viable target in HGSOC., (©2019 American Association for Cancer Research.)

Preeclampsia (PE) is a major cause of maternal mortality and morbidity worldwide. Although there has been great progress in the understanding of PE, the exact cause for the disease development is still unclear. Recently, studies showed that genetic deletion of ELABELA (ELA, also known as APELA) could induce PE-like symptoms in mice. However, the role of ELA in the disease development of PE remains elusive. Our objective was to measure the changes of ELA levels in maternal serum, urine, and placenta from preeclamptic pregnant women and healthy pregnant women and evaluate the correlation between ELA levels and the occurrence of PE. Additionally, we investigated the effect of ELA on the migration and proliferation of human trophoblast cells. ELA levels are significantly decreased in late-onset PE pregnancies compared with normal pregnancies. The mRNA and protein expressions of ELA and the apelin receptor (APLNR or APJ) in late-onset PE placental tissues are also decreased. Furthermore, our in vitro study showed that the addition of ELA significantly increased the invasion ability and proliferation of trophoblast cells, which were inhibited by the APJ-specific antagonist ML221. Our study identified ELA as significantly decreased in late-onset PE; therefore, it might play an important role in the pathogenesis of late-onset PE.

Recent studies have made significant progress in identifying distinct populations of peripheral neurons involved in itch transmission, whereas the cellular identity of spinal interneurons that contribute to itch processing is still a debate. Combining genetic and pharmacological ablation of spinal excitatory neuronal subtypes and behavioral assays, we demonstrate that spinal somatostatin-positive (SOM) excitatory interneurons transmit pruritic sensation. We found that the ablation of spinal SOM/Lbx1 (SOM) neurons caused significant attenuation of scratching responses evoked by various chemical pruritogens (chemical itch). In an attempt to identify substrates of spinal itch neural circuit, we observed that spinal SOM neurons partially overlapped with neurons expressing natriuretic peptide receptor A (Npra), the receptor of peripheral itch transmitter B-type natriuretic peptide. Spinal SOM neurons, however, did not show any overlap with itch transmission neurons expressing gastrin-releasing peptide receptor in the dorsal spinal cord, and the gastrin-releasing peptide-triggered scratching responses were intact after ablating spinal SOM neurons. Dual ablation of SOM and Npra neurons in the spinal cord reduced chemical itch responses to a greater extent than ablation of SOM or Npra neurons alone, suggesting the existence of parallel spinal pathways transmitting chemical itch. Furthermore, we showed that SOM peptide modulated itch processing through disinhibition of somatostatin receptor 2A-positive inhibitory interneuron. Together, our findings reveal a novel spinal mechanism for sensory encoding of itch perception.

Background: Identification of unknown chemical entities is a major challenge in metabolomics. To address this challenge, we developed a comprehensive targeted profiling strategy, combining 3 complementary liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) techniques and in-house accurate mass retention time (AMRT) databases established from commercial standards. This strategy was used to evaluate the effect of nitisinone on the urinary metabolome of patients and mice with alkaptonuria (AKU). Because hypertyrosinemia is a known consequence of nitisinone therapy, we investigated the wider metabolic consequences beyond hypertyrosinemia., Methods: A total of 619 standards (molecular weight, 45-1354 Da) covering a range of primary metabolic pathways were analyzed using 3 liquid chromatography methods-2 reversed phase and 1 normal phase-coupled to QTOF-MS. Separate AMRT databases were generated for the 3 methods, comprising chemical name, formula, theoretical accurate mass, and measured retention time. Databases were used to identify chemical entities acquired from nontargeted analysis of AKU urine: match window theoretical accurate mass ±10 ppm and retention time ±0.3 min., Results: Application of the AMRT databases to data acquired from analysis of urine from 25 patients with AKU (pretreatment and after 3, 12, and 24 months on nitisinone) and 18 HGD -/- mice (pretreatment and after 1 week on nitisinone) revealed 31 previously unreported statistically significant changes in metabolite patterns and abundance, indicating alterations to tyrosine, tryptophan, and purine metabolism after nitisinone administration., Conclusions: The comprehensive targeted profiling strategy described here has the potential of enabling discovery of novel pathways associated with pathogenesis and management of AKU., (© 2019 American Association for Clinical Chemistry.)

Background: Tuberculosis (TB) is India's major public health problem. According to the WHO, India harbors the largest number of cases, and TB control remains a challenge in diagnosis, drug resistance, and treatment. We undertook this study to compare the isolation rates of Mycobacterium tuberculosis (MTB) in agar, egg-based media, incidence of multidrug-resistant (MDR), and extended drug resistance (XDR) in MTB. This study aimed to compare and evaluate thin-layered agar (TLA) for the cultivation and drug susceptibility pattern of MTB with the conventional egg-based Lowenstein-Jensen's (LJ) medium and to differentiate atypical Mycobacterium by incorporating para-nitrobenzoic acid (PNB) in the TLA medium. This cross-sectional study was conducted in Sri Ramachandra Medical College and Research Institute, Porur, Chennai., Methods: A total of 68 smear-positive samples were inoculated into TLA (Middle Brook 7H 11) and LJ media with and without antibiotics (rifampicin, isoniazid, and ofloxacin) simultaneously after decontamination by the modified Petroff's method. TLA with PNB was also used to differentiate the growth of nontuberculous mycobacterium (NTM). Incubation was done at 37°C, and reading was taken every 3 rd day for 6 weeks in case of TLA and for 8 weeks in case of LJ medium., Results: Out of the 68 samples, 64 (94.1%) grew in LJ, and the growth observed at the end of the 1 st , 2 nd , 3 rd , 4 th , 5 th , and 6-10 th weeks was 0, 12 (18.8%), 10 (15.6%), 14 (21.9%), 15 (23.4%), and 13 (20.3%), respectively. Similarly, in TLA, 65 (95.5%) samples were grown, among which 22 (33.8%) grew in the 1 st week and the rest (43 [66.2%]) in the 2 nd week. MDR and XDR were observed in 4 (5.8%) and 3 (4.4%) samples, respectively. Seven of them were NTM., Conclusions: TLA is a better medium, with time to positivity ranging from 1 to 2 weeks with drug susceptibility and the pattern is also comparable with LJ medium. Incorporation of PNB in TLA helps in differentiating NTM., Competing Interests: None

Involved in the tyrosine degradation pathway, 4-hydroxyphenylpyruvate dioxygenase (HPPD) is an important target for treating type I tyrosinemia. To discover novel HPPD inhibitors, we proposed a hydrophobicity-oriented drug design (HODD) strategy based on the interactions between HPPD and the commercial drug NTBC. Most of the new compounds showed improved activity, compound d23 being the most active candidate (IC 50  = 0.047 μM) with about 2-fold more potent than NTBC (IC 50  = 0.085 μM). Therefore, compound d23 is a potential drug candidate to treat type I tyrosinemia., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Leishmaniasis is a neglected disease caused by a trypanosomatid protozoan of the genus Leishmania . Most drugs used to treat leishmaniasis are highly toxic, and the emergence of drug-resistant strains has been observed. Therefore, new therapeutic targets against leishmaniasis are required. Several isoprenoid compounds, including dolichols or ubiquinones, have been shown to be important for cell viability and proliferation in various trypanosomatid species. Here, we detected the biosynthesis of tocopherol in Leishmania ( L .) amazonensis promastigotes in vitro through metabolic labelling with [1-(n)-3H]-phytol. Subsequently, we confirmed the presence of vitamin E in the parasite by gas chromatography-mass spectrometry. Treatment with usnic acid or nitisinone, inhibitors of precursors of vitamin E synthesis, inhibited growth of the parasite in a concentration-dependent manner. This study provides the first evidence of tocopherol biosynthesis in a trypanosomatid and suggests that inhibitors of the enzyme 4-hydroxyphenylpyruvate dioxygenase may be suitable for use as antileishmanial compounds., Database: The amino acid sequence of a conserved hypothetical protein [Leishmania mexicana MHOM/GT/2001/U1103] has been deposited in GenBank (CBZ28005.1)., Competing Interests: The authors declare no conflict of interest.

Trans-sialidase of Trypanosoma cruzi (TcTS) is a key enzyme in the infection process from parasite to host; therefore, it has been considered an important target for developing new anti-Chagas drugs. Different compounds with trypanocidal activity and/or inhibition of TcTS have been reported; however, some benzoic acid derivatives have shown high enzymatic inhibition but low trypanocidal activity and viceversa. These results show that each compound may possess a different mechanism of action. Based on the above, the compound 4-amino-3-nitrobenzoic acid (16), a potent TcTS inhibitor (77% inhibition in enzymatic assays) was selected to evaluate its effects on the expression level of the TS gene in T. cruzi epimastigotes and determine its involvement in the mechanism of action. Results showed an increase in the expression level of the TcTS gene, which confirmed that compound 16, has a direct effect on TcTS.

Purpose: Nitisinone inhibits the cytochrome P450 (CYP) subfamilies CYP2C9, CYP2D6, and CYP2E1 and the organic anion transporter (OAT) isoforms OAT1 and OAT3 in vitro. Since the effect of nitisinone on these enzymes and transporters in humans is still unknown, the purpose of this study was to evaluate the effect of nitisinone on these CYP subfamilies and OAT isoforms., Methods: This was an open-label, nonrandomized, two-arm, phase 1 study (EudraCT: 2016-004297-17) in healthy volunteers. The substrates (tolbutamide, metoprolol, and chlorzoxazone for the respective CYPs and furosemide for the OATs) were administered as single doses, before and after 15 days of once daily dosing of 80 mg nitisinone, to determine the AUC ∞ ratios ([substrate+nitisinone]/[substrate]). Nitisinone pharmacokinetics, safety, and tolerability were also assessed, and blood and urine were collected to determine substrate and nitisinone concentrations by LC-MS/MS., Results: Thirty-six subjects were enrolled with 18 subjects included in each arm. The least square mean ratio (90% confidence interval) for AUC ∞ was 2.31 (2.11-2.53) for tolbutamide, 0.95 (0.88-1.03) for metoprolol, 0.73 (0.67-0.80) for chlorzoxazone, and 1.72 (1.63-1.81) for furosemide. Clinically relevant nitisinone steady-state concentrations were reached after 12 days: mean C av,ss of 94.08 μM. All treatments were well tolerated, and no safety concerns were identified., Conclusions: Nitisinone did not affect CYP2D6 activity, was a weak inducer of CYP2E1, and was a weak inhibitor of OAT1 and OAT3. Nitisinone was a moderate inhibitor of CYP2C9, and treatment may therefore result in increased plasma concentrations of comedications metabolized primarily via this enzyme., Clinical Trial Registry Identification: EudraCT 2016-004297-17.

When treating glaucoma, excessive scar tissue reactions reduce the postoperative survival rate of the filtering bleb. Accumulating evidence has demonstrated that the proliferation, migration and extracellular matrix (ECM) synthesis of fibroblasts are important molecular mechanisms underlying scar formation. Recent evidence has demonstrated that chloride channels play an important role in controlling cell proliferation, apoptosis, migration and the cell cycle process in several cell types, but the effects of chloride channels on conjunctival fibroblasts have not be studied. The aim of the present study was to investigate the effects of the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) on cell proliferation, apoptosis, migration, cell cycle progression and ECM synthesis in human conjunctival fibroblasts (HConFs), and to further investigate the mechanism of resistance to scar formation following glaucoma filtration surgery. HConFs were exposed to NPPB or lubiprostone. Cell proliferation and viability was evaluated using the Cell Counting Kit-8. Cell migration was measured using Transwell migration and scratch‑wound assays. Flow cytometry was used to study apoptosis and cell cycle progression. Quantitative polymerase chain reaction and western blot analyses were performed to determine mRNA and protein expression levels, respectively. Following NPPB treatment, HConFs exhibited reduced proliferation and migration, along with increased apoptosis. NPPB also inhibited cell cycle progression by arresting cells in the G0̸G1 phase and reducing collagen I and fibronectin expression, as well as the phosphorylation of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT). However, lubiprostone treatment exerted the opposite effects on HConFs. Therefore, NPPB treatment inhibited proliferation, migration, cell cycle progression and synthesis of the ECM, while promoting apoptosis in HConFs, by inhibiting the PI3K̸AKT signaling pathway.

Background/aims: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner., Methods: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (Vmem) and swelling-dependent chloride (Cl-) currents (IClswell, VSOR, VRAC) were performed using the whole-cell patch clamp technique., Results: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl- current under isotonic conditions resembling features of the swelling-activated Cl- current (IClswell). The cell membrane potential (Vmem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na+-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells., Conclusion: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Human red blood cells infected with the malaria parasite Plasmodium falciparum show an increased permeability to a number of solutes. We have previously demonstrated that such infected cells take up glutamate via a member of the excitatory amino acid transporter protein family (EAAT), namely EAAT3. Babesia divergens is a parasite that also infects human erythrocytes, and also induces increased solute permeability, including for glutamate. Here we have investigated whether glutamate uptake in B. divergens infected human red blood cells is also dependent on EAAT3 activity. We find that, although B. divergens infected cells do take up glutamate, this uptake is independent on EAAT3. Thus, though infecting the same host cell, two related parasites have developed distinct pathways to obtain access to nutrients from the extracellular milieu., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Background/aims: In the human genome, more than 400 genes encode ion channels, which are ubiquitously expressed and often coexist and participate in almost all physiological processes. Therefore, ion channel blockers represent fundamental tools in discriminating the contribution of individual channel types to a physiological phenomenon. However, unspecific effects of these compounds may represent a confounding factor. Three commonly used chloride channel inhibitors, i.e. 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS), 5-nitro-2-[(3-phenylpropyl) amino]benzoic acid (NPPB) and the anti-inflammatory drug niflumic acid were tested to identify the lowest concentration effective on Cl- channels and ineffective on K+ channels., Methods: The activity of the above mentioned compounds was tested by whole cell patch-clamp on the swelling-activated Cl- current ICl,swell and on the endogenous voltage-dependent, outwardly rectifying K+ selective current in human kidney cell lines (HEK 293/HEK 293 Phoenix)., Results: Micromolar (1-10 µM) concentrations of DIDS and NPPB could not discriminate between the Cl- and K+ selective currents. Specifically, 1 µM DIDS only affected the K+ current and 10 µM NPPB equally affected the Cl- and K+ currents. Only relatively high (0.1-1 mM) concentrations of DIDS and prolonged (5 minutes) exposure to 0.1-1 mM NPPB preferentially suppressed the Cl- current. Niflumic acid preferentially inhibited the Cl- current, but also significantly affected the K+ current. The endogenous voltage-dependent, outwardly rectifying K+ selective current in HEK 293/HEK 293 Phoenix cells was shown to arise from the Kv 3.1 channel, which is extensively expressed in brain and is involved in neurological diseases., Conclusion: The results of the present study underscore that sensitivity of a given physiological phenomenon to the Cl- channel inhibitors NPPB, DIDS and niflumic acid may actually arise from an inhibition of Cl- channels but can also result from an inhibition of voltage-dependent K+ channels, including the Kv 3.1 channel. The use of niflumic acid as anti-inflammatory drug in patients with concomitant Kv 3.1 dysfunction may result contraindicated., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The mechanism of brain edema is complex and still remains unclear. Our aim was to investigate the regional differences of cell volume and intracellular Ca 2+ concentration ([Ca 2+ ] i ) dynamics during hypotonic stress in male mouse hemi-brain slices. Brain slices were loaded with the fluorescence Ca 2+ indicator fura-2, and cell volume and [Ca 2+ ] i in the lateral cerebral cortex (LCC) and hippocampal CA1 (CA1) region were measured simultaneously during exposure to hypotonic stress using Ca 2+ insensitive (F360) and Ca 2+ sensitive fluorescence (F380), respectively. Brain cell swelling induced by hypotonic stress was followed by a regulatory volume change that coincided with an increase in [Ca 2+ ] i . The degrees of change in cell volume and [Ca 2+ ] i were significantly different between the LCC and CA1. The increase in cell volume and [Ca 2+ ] i in the LCC, but not in the CA1, was decreased by the transient receptor potential channel blockers LaCl 3 and GdCl 3 . The increase in [Ca 2+ ] i in both the LCC and CA1, was significantly decreased by the intracellular Ca 2+ modulators thapsigargin and xestospongin C. The K + channel activator isoflurane and Cl - channel blocker NPPB significantly decreased [Ca 2+ ] i in the LCC. This study demonstrated that, between cells located in the LCC and in the CA1, the characteristics of brain edema induced by hypotonic stress are different. This can be ascribed to the different contribution of volume sensitive G-protein coupled receptor and stretch sensitive Ca 2+ channels., (© 2017 Wiley Periodicals, Inc.)

Purpose: Ctn[15-34], a carboxyl-terminal fragment of crotalicidin (a cathelicidin from the venom gland of a South American rattlesnake), has shown antifungal activity against clinical and standard strains of Candida species. The aim of the present work was to investigate the underlying mechanisms of the candidicidal activity of Ctn[15-34]., Methodology: The time-kill profile and drug synergism were evaluated by means of a microdilution assay and multi-parametric flow cytometry. The presumptive interaction of Ctn[15-34] with lipid membranes was estimated in vitro with a lipid-mimic compound, the chromogenic substance 4-nitro-3-(octanoyloxy)benzoic acid (4N3OBA).Results/Key findings. The absorbance increment (at 425 nm) indicated a concentration- and time-dependent in-solution association between Ctn[15-34] and 4N3OBA. The interaction of Ctn[15-34] with Candida cells was confirmed by flow cytometric measurements with the 5(6)-carboxyfluorescein-labelled peptide (CF-Ctn[15-34]). Analysis of the killing time of Candida exposed to Ctn[15-34] and amphotericin B (AMB) showed that both the peptide and polyene drug reduce the number of c.f.u. but in mechanistically different ways. The Ctn[15-34] peptide alone caused yeast cell membrane disruption, which was confirmed by lactate dehydrogenase leakage and biomarkers of cell death mediated by necrosis., Conclusion: Overall, Ctn[15-34] displays a synergistic antifungal activity with AMB, an effect that can be further developed into a multi-target therapeutic option with other antimycotics currently in use.

Pathological retinal angiogenesis is caused by the progression of ischemic retinal diseases and can result in retinal detachment and irreversible blindness. This neovascularization is initiated from the retinal veins and their associated capillaries and involves the overgrowth of vascular endothelial cells. Since expression of the apelin receptor (APJ) is restricted to the veins and proliferative endothelial cells during physiological retinal angiogenesis, in the present study, we investigated the effect of APJ inhibition on pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy (OIR). In vitro experiments revealed that ML221, an APJ antagonist, suppressed cultured-endothelial cell proliferation in a dose-dependent manner. Intraperitoneal administration of ML221 inhibited pathological angiogenesis but enhanced the recovery of normal vessels into the ischemic regions in the retina of the OIR model mice. ML221 did not affect the expression levels of vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) in the retina. APJ was highly expressed in the endothelial cells within abnormal vessels but was only detected in small amounts in morphologically normal vessels. These results suggest that APJ inhibitors selectively prevent pathological retinal angiogenesis and that the drugs targeting APJ may be new a candidate for treating ischemic retinopathy.

Anion channels and connexin hemichannels are permeable to amino acid neurotransmitters. It is hypothesized that these conductive pathways release GABA, thereby influencing ambient GABA levels and tonic GABAergic inhibition. To investigate this, we measured the effects of anion channel/hemichannel antagonists on tonic GABA currents of rat hippocampal neurons. In contrast to predictions, blockade of anion channels and hemichannels with NPPB potentiated tonic GABA currents of neurons in culture and acute hippocampal slices. In contrast, the anion channel/hemichannel antagonist carbenoxolone (CBX) inhibited tonic currents. These findings could result from alterations of ambient GABA concentration or direct effects on GABA A receptors. To test for effects on GABA A receptors, we measured currents evoked by exogenous GABA. Coapplication of NPPB with GABA potentiated GABA-evoked currents. CBX dose-dependently inhibited GABA-evoked currents. These results are consistent with direct effects of NPPB and CBX on GABA A receptors. GABA release from hippocampal cell cultures was directly measured using HPLC. Inhibition of anion channels with NPPB or CBX did not affect GABA release from cultured hippocampal neurons. NPPB reduced GABA release from pure astrocytic cultures by 21%, but the total GABA release from astrocytes was small compared to that of mixed cultures. These data indicate that drugs commonly used to antagonize anion channels and connexin hemichannels may affect tonic currents via direct effects on GABA A receptors and have negligible effects on ambient GABA concentrations. Interpretation of experiments using NPPB or CBX should include consideration of their effects on tonic GABA currents.

Human spermatozoa encounter an osmotic decrease from 330 to 290 mOsm l-1 when passing through the female reproductive tract. We aimed to evaluate the role of chloride channels in volume regulation and sperm motility from patients with asthenozoospermia. Spermatozoa were purified using Percoll density gradients. Sperm volume was measured as the forward scatter signal using flow cytometry. Sperm motility was analyzed using computer-aided sperm analysis (CASA). When transferred from an isotonic solution (330 mOsm l-1 ) to a hypotonic solution (290 mOsm l-1 ), cell volume was not changed in spermatozoa from normozoospermic men; but increased in those from asthenozoospermic samples. The addition of the chloride channel blockers, 4,4'-diisothiocyanatostilbene-2,2'- isulfonic acid (DIDS) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) to the hypotonic solution caused the normal spermatozoa to swell but did not increase the volume of those from the asthenozoospermic semen. DIDS and NPPB decreased sperm motility in both sets of semen samples. The inhibitory effect of NPPB on normal sperm motility was much stronger than on spermatozoa from the asthenozoospermic samples. Both sperm types expressed ClC-3 chloride channels, but the expression levels in the asthenozoospermic samples were much lower, especially in the neck and mid-piece areas. Spermatozoa from men with asthenozoospermia demonstrated lower volume regulating capacity, mobility, and ClC-3 expression levels (especially in the neck) than did normal spermatozoa. Thus, chloride channels play important roles in the regulation of sperm volume and motility and are downregulated in cases of asthenozoospermia.

Purpose: Cholangiocarcinoma (CCA) is a malignancy of the biliary epithelium that is associated with low five-year survival. The apelin receptor (APLNR), which is activated by the apelin peptide, has not been studied in CCA. The purpose of this study is to determine if inhibition of the apelin/APLNR axis can inhibit CCA growth., Methods: Immunohistochemistry, rtPCR, immunofluorescence, flow cytometry, and ELISA was used to measure APLNR expression in human CCA cells and tissues. Mz-ChA-1 cells were treated with increasing concentrations of apelin and ML221, an APLNR antagonist. Expression of proliferative and angiogenic genes were measured via rtPCR. In vivo, Mz-ChA-1 cells were injected into the flanks of nu/nu mice, which were treated with ML221 (150 μg/kg) via tail vein injection., Results: Expression of the apelin/APLNR axis was increased in CCA. In vitro, CCA proliferation and angiogenesis was inhibited by ML221 treatment. ML221 treatment significantly decreased tumor growth in nu/nu mice., Conclusion: The apelin/APLNR axis regulates CCA proliferation and angiogenesis. Inhibition of the apelin/APLNR axis decreases tumor growth in our xenograft model. Targeting APLNR signaling has the potential to serve as a novel, tumor directed therapy for CCA., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

A series of novel cholinesterase inhibitors containing nitrobenzoate core structure were synthesized by a facile and efficient method. The structure of the novel compounds were fully characterized and confirmed by analytical as well as spectroscopic methods. Compound indicated as 2f was found to possess the best cholinesterase inhibitory activities of all the evaluated compounds. Results suggest that 2f is a selective acetylcholinesterase inhibitor, although it also inhibits butyrylcholinesterase at higher concentration. Kinetics inhibition result suggest that 2f is a mixed-mode inhibitor of acetylcholinesterase. In addition, it was found to have low cytotoxicity. Molecular docking on compound 2f was carried out to rationalize the observed in vitro enzymatic assay results. Most importantly, the potential of nitrobenzoate derivatives as cholinesterase inhibitor was shown through this study. In summary, we discovered nitrobenzoates as a new scaffold that may eventually yield useful compounds in treatment of Alzheimer's disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This chapter provides a clinical perspective on the challenges that stand between current clinical practice and a cure for hepatorenal tyrosinemia (HT1). HT1 has been transformed in the last 50 years from an aggressive often undiagnosed childhood disease causing liver failure or liver cancer, with infant death in most patients, to a condition that is detectable at birth, and for which treatment with nitisinone (NTBC) and diet can prevent detectable liver or kidney abnormalities. What challenges remain? The properties of the affected metabolic pathway and the broad spectrum of severity seen in untreated patients are incompletely understood but potentially important for patients. Available treatments have potential complications, including liver transplantation (risks of surgery and of immunosuppression to prevent rejection), nitisinone and diet therapy (hypertyrosinemia, corneal opacities, nutritional imbalances and possibly developmental delay). The detection of liver cancer is imperfect and laborious. The effects of tyrosinemia during pregnancy are little-known. Although animal models of HT1 are becoming standard research tools in cell replacement and gene modification therapy, these techniques are not currently applicable to HT1 itself. Treatment adherence is variable, causing concern about long term outcome for some patients. Around the world, there are great disparities in the diagnosis and treatment of HT1. Most affected individuals are born in places where newborn screening for HT1 is not performed and where appropriate treatment is not available. We hope that this list will help to focus on some of these remaining obstacles to a cure for HT1.

The discovery that a natural product leptospermone had herbicidal activity formed the starting point for chemical synthesis to find more activity and selectivity. A series of molecules called triketones were found to possess good activity and 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC) was selected for toxicology testing. NTBC fed at low doses to rats and dogs caused keratopathy, which on cessation of the diet recovered. Mice, rabbits and monkeys fed NTBC did not show this response. Research discovered that NTBC caused tyrosinaemia which was due to inhibition of the enzyme 4-hydroxyphenylpyruvate dioxygenase in both mammals and plants thereby finding a novel target for killing plants. NTBC was also used sucessfully as a drug to treat a rare inborn error of metabolism, tyrosinaemia type I, in collaboration with Professor's Sven Lindstedt and Elisabeth Holme. Understanding the mechanism of toxicity of NTBC led to novel herbicide discovery and saved the lives of children with acute tyrosinaemia type I.

Hereditary tyrosinemia type 1 (HT1) is characterized by severe progressive liver disease and renal tubular dysfunction. Kidney involvement is characterized by hypophosphatemic rickets and Fanconi syndrome. Different animal models were useful to investigate the pathophysiology of the disease and the effects of NTBC therapy on liver and kidney function. NTBC has revolutionized the prognosis of HT1 and its acute and chronic effects on renal tubular function have been proved, with normalization of tubular function within a few weeks, particularly hypophosphatemia and proteinuria. NTBC therapy is highly effective in improving renal function both at short and long-term. However, its efficacy critically depends on the age at start of treatment with normal outcome in patients diagnosed at birth by newborn screening.

Untreated HT1 rapidly degenerates into very severe liver complications often resulting in liver cancer. The molecular basis of the pathogenic process in HT1 is still unclear. The murine model of FAH-deficiency is a suitable animal model, which represents all phenotypic and biochemical manifestations of the human disease on an accelerated time scale. After removal of the drug 2-(2-N-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), numerous signaling pathways involved in cell proliferation, differentiation and cancer are rapidly deregulated in FAH deficient mice. Among these, the Endoplasmic reticulum (ER) pathway, the heat stress response (HSR), the Nrf2, MEK and ERK pathways, are highly represented. The p21 and mTOR pathways critical regulators of proliferation and tumorigenesis have also been found to be dysregulated. The changes in these pathways are described and related to the development of liver cancer.

Tyrosinemia type I is a recessive inborn error of metabolism caused by mutations in the fumarylacetoacetate hydrolase (FAH) gene, coding for the final enzyme in the metabolism of tyrosine. This renders FAH nonfunctional and without treatment, toxic metabolites accumulate causing liver and kidney damage. Introduction of the drug NTBC in 2002 offered a treatment which inhibits an upstream enzyme, preventing the production of the toxic metabolites. There is now a long-term survival rate of greater than 90 % in children, but there are reports of lower cognitive function and IQ as well as schooling and behavioral problems in these children. We studied a mouse model of tyrosinemia type I to gain insight into the effects of tyrosinemia type I and treatment with NTBC on mouse learning, memory, and behavior. In the Barnes maze, visual and spatial cues can be used by mice to remember the location of a dark escape box. The primary time, distance, and strategy taken by the mice to locate the escape box is a measure of learning and memory. Our findings show that mice with tyrosinemia type I were slower to learn than wild-type mice treated with NTBC and made more mistakes, but were capable of learning and storing long-term memory. After learning the location of the target hole, mice with tyrosinemia type I respond differently to a change in location and were less flexible in learning the new target hole location. Our findings suggest that this slower learning and cognitive difference is caused by tyrosinemia type I and not by the treatment with NTBC.

Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated but ATP-gated chloride channel. Previous studies suggested that VX-770 [ivacaftor, N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide], a CFTR potentiator now used in clinics, increases the open probability of CFTR by shifting the gating conformational changes to favor the open channel configuration. Recently the chloride channel blocker and CFTR potentiator 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) has been reported to enhance CFTR activity by a mechanism that exploits the ATP hydrolysis-driven, nonequilibrium gating mechanism unique to CFTR. Surprisingly however, NPPB increased the activity of nonhydrolytic G551D-CFTR, the third most common disease-associated mutation. Here, we further investigated the mechanism of NPPB's effects on CFTR gating by assessing its interaction with well-studied VX-770. Interestingly, once G551D-CFTR was maximally potentiated by VX-770, NPPB further increased its activity. However, quantitative analysis of this drug-drug interaction suggests that this pharmacologic synergism is not due to independent actions of NPPB and VX-770 on CFTR gating; instead, our data support a dependent mechanism involving two distinct binding sites. This latter idea is further supported by the observation that the locked-open time of a hydrolysis-deficient mutant K1250A was shortened by NPPB but prolonged by VX-770. In addition, the effectiveness of NPPB, but not of VX-770, was greatly diminished in a mutant whose second nucleotide-binding domain was completely removed. Interpreting these results under the framework of current understanding of CFTR gating not only reveals insights into the mechanism of action for different CFTR potentiators but also brings us one step forward to a more complete schematic for CFTR gating., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Multidrug resistance is a major limitation for microtubule-binding agents in cancer treatment. Here we report a novel microtubule inhibitor (2-morpholin-4-yl-5-nitro-benzoic acid 4-methylsulfanyl-benzyl ester, IMB5046), its cytotoxicity against multidrug-resistant cell lines and its antitumor efficacy in animal models. IMB5046 disrupted microtubule structures in cells and inhibited purified tubulin polymerization in vitro. It bound to the colchicine pocket of tubulin. IMB5046 displayed potent cytotoxicity against multiple tumor cell lines with an IC50 range of 0.037-0.426 μM. Notably, several multidrug-resistant cell lines which were resistant to colchicine, vincristine and paclitaxel remained sensitive to IMB5046. IMB5046 was not a P-glycoprotein substrate. IMB5046 blocked cell cycle at G2/M phase and induced cell apoptosis. Microarray assay indicated that the differentially expressed genes after IMB5046 treatment were highly related to immune system, cell death and cancer. In a mouse xenograft model IMB5046 inhibited the growth of human lung tumor xenograft by 83% at a well-tolerated dose. It is concluded that IMB5046 is a tubulin polymerization inhibitor with novel chemical structure and can overcome multidrug resistance. It is a promising lead compound for cancer chemotherapy, especially for treatment of multidrug-resistant tumors.

VX-770 (Ivacaftor) has been approved for clinical usage in cystic fibrosis patients with several CFTR mutations. Yet the binding site(s) on CFTR for this compound and other small molecule potentiators are unknown. We hypothesize that insight into this question could be gained by comparing the effect of potentiators on CFTR channels from different origins, e.g., human, mouse, and Xenopus (frog). In the present study, we combined this comparative molecular pharmacology approach with that of computer-aided drug discovery to identify and characterize new potentiators of CFTR and to explore possible mechanism of action. Our results demonstrate that 1) VX-770, NPPB, GlyH-101, P1, P2, and P3 all exhibited ortholog-specific behavior in that they potentiated hCFTR, mCFTR, and xCFTR with different efficacies; 2) P1, P2, and P3 potentiated hCFTR in excised macropatches in a manner dependent on the degree of PKA-mediated stimulation; 3) P1 and P2 did not have additive effects, suggesting that these compounds might share binding sites. Also 4) using a pharmacophore modeling approach, we identified three new potentiators (IOWH-032, OSSK-2, and OSSK-3) that have structures similar to GlyH-101 and that also exhibit ortholog-specific potentiation of CFTR. These could potentially serve as lead compounds for development of new drugs for the treatment of cystic fibrosis. The ortholog-specific behavior of these compounds suggest that a comparative pharmacology approach, using cross-ortholog chimeras, may be useful for identification of binding sites on human CFTR., (Copyright © 2016 the American Physiological Society.)

We tested the hypothesis that ex vivo hepatocyte gene therapy can correct the metabolic disorder in fumarylacetoacetate hydrolase-deficient (Fah(-/-)) pigs, a large animal model of hereditary tyrosinemia type 1 (HT1). Recipient Fah(-/-) pigs underwent partial liver resection and hepatocyte isolation by collagenase digestion. Hepatocytes were transduced with one or both of the lentiviral vectors expressing the therapeutic Fah and the reporter sodium-iodide symporter (Nis) genes under control of the thyroxine-binding globulin promoter. Pigs received autologous transplants of hepatocytes by portal vein infusion. After transplantation, the protective drug 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC) was withheld from recipient pigs to provide a selective advantage for expansion of corrected FAH(+) cells. Proliferation of transplanted cells, assessed by both immunohistochemistry and noninvasive positron emission tomography imaging of NIS-labeled cells, demonstrated near-complete liver repopulation by gene-corrected cells. Tyrosine and succinylacetone levels improved to within normal range, demonstrating complete correction of tyrosine metabolism. In addition, repopulation of the Fah(-/-) liver with transplanted cells inhibited the onset of severe fibrosis, a characteristic of nontransplanted Fah(-/-) pigs. This study demonstrates correction of disease in a pig model of metabolic liver disease by ex vivo gene therapy. To date, ex vivo gene therapy has only been successful in small animal models. We conclude that further exploration of ex vivo hepatocyte genetic correction is warranted for clinical use., (Copyright © 2016, American Association for the Advancement of Science.)