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Since the middle of the twentieth century, the use of dyes has become more common in every food group as well as in the pharmaceutical, textile and cosmetic industries. Azo dyes, including carmoisine, are the most important of the dye classes with the widest color range. In this study, the effects of carmoisine exposure on the embryonic development of zebrafish at a wide dose scale, including recommended and overexposure doses (from 4 to 2000 ppm), were investigated in detail. For this purpose, many morphological and physiological parameters were examined in zebrafish exposed to carmoisine at determined doses for 96 h, and the mechanisms of action of the changes in these parameters were tried to be clarified with the metabolite levels determined. The no observed effect concentration (NOEC) and median lethal concentration (LC 50 ) were recorded at 5 ppm and 1230.53 ppm dose at 96 hpf, respectively. As a result, it was determined that the applied carmoisine caused serious malformations, reduction in height and eye diameter, increase in the number of free oxygen radicals, in apoptotic cells and in lipid accumulation, decrease in locomotor activity depending on the dose and at the highest dose, decrease in blood flow rate. In the metabolome analysis performed to elucidate the metabolism underlying all these changes, 45 annotated metabolites were detected., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Novel pyridine-containing sultones were synthesized and evaluated for their cholinesterase (ChE) inhibitory activity. Most of compounds showed selective acetylcholinesterase (AChE) inhibitory activity. The structure-activity relationship (SAR) showed: (i) the fused pyridine-containing sultones increase AChE inhibition, series B>series A; (ii) for series A, the effect of the 4-substituent on AChE activity, p->m- or o-; (iii) for series B, a halophenyl group increase activity. Compound B4 (4-(4-chlorophenyl)-2,2-dioxide-3,4,5,6-tetrahydro-1,2-oxathiino[5,6-h]quinoline) was identified as a selective AChE inhibitor (IC 50 =8.93 μM), and molecular docking studies revealed a good fit into TcAChE via hydrogen interactions between the δ-pyridylsultone scaffold with Asp72, Ser122, Phe288, Phe290 and Trp84. Compound B4 showed reversible and non-competitive (K i =7.67 μM) AChE inhibition, nontoxicity and neuroprotective activity. In vivo studies confirmed that compound B4 could ameliorate the cognitive performance of scopolamine-treated C57BL/6 J mice, suggesting a significant benefit of AChE inhibition for a disease-modifying treatment of AD., (© 2021 Wiley-VCH GmbH.)

Rhabdomyosarcoma (RMS) is a malignant soft tissue cancer that develops mostly in children and young adults. With regard to histopathology, four rhabdomyosarcoma types are distinguishable: embryonal, alveolar, pleomorphic and spindle/sclerosing. Currently, increased amounts of evidence indicate that not only gene mutations, but also epigenetic modifications may be involved in the development of RMS. Epigenomic changes regulate the chromatin architecture and affect the interaction between DNA strands, histones and chromatin binding proteins, thus, are able to control gene expression. The main aim of the study was to assess the role of protein arginine methyltransferases (PRMT) in the cellular biology of rhabdomyosarcoma. In the study we used two pan-inhibitors of PRMT, called AMI-1 and SAH, and evaluated their effects on proliferation and apoptosis of RMS cells. We observed that AMI-1 and SAH reduce the invasive phenotype of rhabdomyosarcoma cells by decreasing their proliferation rate, cell viability and ability to form cell colonies. In addition, microarray analysis revealed that these inhibitors attenuate the activity of the PI3K-Akt signaling pathway and affect expression of genes related to it.

As a therapeutic approach, epigenetic modifiers have the potential to enhance the efficacy of chemotherapeutic agents. Protein arginine methyltransferase 5 (PRMT5), highly expressed in lung adenocarcinoma, was identified to be involved in tumorigenesis. In the current study, we examined the potential antineoplastic activity of PRMT5 inhibitor, arginine methyltransferase inhibitor 1 (AMI-1), and cisplatin on lung adenocarcinoma. Bioinformatic analyses identified apoptosis, DNA damage, and cell cycle progression as the main PRMT5-associated functional pathways, and survival analysis linked the increased PRMT5 gene expression to worse overall survival in lung adenocarcinoma. Combined AMI-1 and cisplatin treatment significantly reduced cell viability and induced apoptosis. Cell cycle arrest in A549 and DMS 53 cells was evident after AMI-1, and was reinforced after combination treatment. Western blot analysis showed a reduction in demethylation histone 4, a PRMT5- downstream target, after treatment with AMI-1 alone or in combination with cisplatin. While the combination approach tackled lung cancer cell survival, it exhibited cytoprotective abilities on HBEpC (normal epithelial cells). The survival of normal bronchial epithelial cells was not affected by using AMI-1. This study highlights evidence of novel selective antitumor activity of AMI-1 in combination with cisplatin in lung adenocarcinoma cells.

Vascular dysfunction in cardiovascular diseases includes vasomotor response impairments, endothelial cells (ECs) activation, and smooth muscle cells (SMCs) proliferation and migration to the intima. This results in intimal hyperplasia and vessel failure. We previously reported that activation of the P2Y11 receptor (P2Y11R) in human dendritic cells, cardiofibroblasts and cardiomyocytes was protective against hypoxia/reoxygenation (HR) lesions. In this study, we investigated the role of P2Y11R signaling in vascular dysfunction. P2Y11R activity was modulated using its pharmacological agonist NF546 and antagonist NF340. Rat aortic rings were exposed to angiotensin II (AngII) and evaluated for their vasomotor response. The P2Y11R agonist NF546 reduced AngII-induced vascular dysfunction by promoting EC-dependent vasorelaxation, through an increased nitric oxide (NO) bioavailability and reduced AngII-induced H 2 O 2 release; these effects were prevented by the use of the P2Y11R antagonist NF340. Human vascular SMCs and ECs were subjected to AngII or H/R simulation in vitro. P2Y11R agonist modulated vasoactive factors in human ECs, that is, endothelial nitric oxide synthase (eNOS) and endothelin-1, reduced SMC proliferation and prevented the switch towards a synthetic phenotype. H/R and AngII increased ECs secretome-induced SMC proliferation, an effect prevented by P2Y11R activation. Thus, our data suggest that P2Y11R activation may protect blood vessels from HR-/AngII-induced injury and reduce vascular dysfunctions. These results open the way for new vasculoprotective interventions.

Nucleotide-binding oligomerization domain-containing proteins 1 and 2 play important roles in immune system activation. Recently, a shift has occurred due to the emerging knowledge that preventing nucleotide-binding oligomerization domains (NODs) signaling could facilitate the treatment of some cancers, which warrants the search for dual antagonists of NOD1 and NOD2. Herein, we undertook the synthesis and identification of a new class of derivatives of dual NOD1/NOD2 antagonists with novel benzofused five-membered sultams. Compound 14k was finally demonstrated to be the most potent molecule that inhibits both NOD1-and NOD2-stimulated NF-κB and MAPK signaling in vitro and in vivo., 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 Masson SAS. All rights reserved.)

Food coloring is often used as a coloring agent in foods, medicines and cosmetics, and it was reported to have certain carcinogenic and mutagenic effects in living organisms. Investigation of physiological parameters using zebrafish is a promising methodology to understand disease biology and drug toxicity for various drug discovery on humans. Zebrafish (Danio rerio) is a well-acknowledged model organism with combining assets such as body transparency, small size, low cost of cultivation, and high genetic homology with humans and is used as a specimen tool for the in-vivo throughput screening approach. In addition, recent advances in microfluidics show a promising alternative for zebrafish manipulation in terms of drug administration and extensive imaging capability. This pilot work highlighted the design and development of a microfluidic detection platform for zebrafish larvae through investigating the effects of food coloring on cardiovascular functionality and pectoral fin swing ability. The zebrafish embryos were exposed to the Cochineal Red and Brilliant Blue FCF pigment solution in a concentration of (0.02‰, 0.2‰) cultured in the laboratory from the embryo stage to hatching and development until 9 days post fertilization (d.p.f.). In addition, zebrafish swimming behaviors in terms of pectoral fin beating towards the toxicity screening were further studied by visualizing the induced flow field. It was evidenced that Cochineal Red pigment at a concentration of 0.2‰ not only significantly affected the zebrafish pectoral fin swing behavior, but also significantly increased the heart rate of juvenile fish. The higher concentration of Brilliant Blue FCF pigment (0.2%) increased heart rate during early embryonic stages of zebrafish. However, zebrafish exposed to food coloring did not show any significant changes in cardiac output. The applications of this proposed platform can be further extended towards observing the neurobiological/hydrodynamic behaviors of zebrafish larvae for practical applications in drug tests.

Glial cell line-derived neurotrophic factor (GDNF) binds the GFR α 1 receptor, and the GDNF-GFR α 1 complex binds to and activates the transmembrane RET tyrosine kinase to signal through intracellular Akt/Erk pathways. To dissect the GDNF-GFR α 1-RET signaling complex, agents that bind and activate RET directly and independently of GFR α 1 expression are valuable tools. In a focused naphthalenesulfonic acid library from the National Cancer Institute database, we identified small molecules that are genuine ligands binding to the RET extracellular domain. These ligands activate RET tyrosine kinase and afford trophic signals irrespective of GFR α 1 coexpression. However, RET activation by these ligands is constrained by GFR α 1, likely via an allosteric mechanism that can be overcome by increasing RET ligand concentration. In a mouse model of retinitis pigmentosa, monotherapy with a small-molecule RET agonist activates survival signals and reduces neuronal death significantly better than GDNF, suggesting therapeutic potential. SIGNIFICANCE STATEMENT: A genuine ligand of RET receptor ectodomain was identified, which acts as an agonist. Binding and agonism are independent of a coreceptor glial cell line-derived neurotrophic factor family receptor α, which is required by the natural growth factor glial cell line-derived neurotrophic factor, and are selective for cells expressing RET. The lead agent protects neurons from death in vivo. This work validates RET receptor as a druggable therapeutic target and provides for potential leads to evaluate in neurodegenerative states. We also report problems that arise when screening chemical libraries., Competing Interests: Patent applications have been filed on behalf of authors (H.U.S., S.J., M.S., and Y.S.)., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Protein arginine methyltransferase 1 (PRMT1), which primarily causes asymmetric arginine methylation of histone and nonhistone proteins, has been found to activate gene expression and mediate multiple pathological processes. Its role in renal fibrosis, however, remains unclear. In the present study, we observed that PRMT1 and its specific epigenetic marker, asymmetric di-methylated histone 4 arginine 3 (H4R3Me2a), were highly expressed in cultured renal interstitial fibroblasts. Treatment of PRMT1 with AMI-1, a selective inhibitor of PRMT1, or silencing PRMT1 with siRNA inhibited serum-induced and transforming growth factor (TGF)-β 1 -induced expression of α-smooth muscle actin (α-SMA) and collagen type I, two hallmarks of renal fibroblast activation, in a dose-dependent and time-dependent manner. In a murine model of renal fibrosis induced by unilateral ureteral obstruction, PRMT1 expression and H4R3Me2a were also upregulated, which was coincident with increased expression of α-SMA, collagen type I, and fibronectin. Administration of AMI-1 reduced PRMT1 and H4R3Me2a expression, attenuated extracellular matrix protein deposition, and inhibited renal fibroblast activation and proliferation. Moreover, AMI-1 treatment inhibited Smad3 phosphorylation and TGF-β receptor I expression but prevented Smad7 downregulation both in the kidney after unilateral ureteral obstruction injury and in cultured renal interstitial fibroblasts exposed to TGF-β 1 . Collectively, these results demonstrate that PRMT1 may mediate renal fibroblast activation and renal fibrosis development through activation of the TGF-β/Smad3 signaling pathway. They also suggest that PRMT1 inhibition may be a potential therapeutic approach for the treatment of fibrotic kidney disease.

Protein fibrous aggregation is associated with many neurodegenerative diseases including Alzheimer's and Parkinson's diseases. To modulate the process, a number of fibrillation inhibitors have been reported, although their working mechanism remains vague, calling for new means to decipher their interaction. Herein, we identified and characterized a novel inhibitor called Crocein Orange G (COG), which inhibited the nucleation and impeded the protofibril formation, revealed by various experimental approaches as well as molecular docking. In particular, the surface-enhanced Raman spectroscopy (SERS) helps to identify the binding sites and illustrate the interaction mechanism and fibrillation process by using Ag IMNPs as SERS substrate for a label-free detection. Combining with molecular docking, the SERS-based approach provides structural information concerning protein-ligand interaction and protein fibrillation. This study suggests that SERS can be a powerful new means to study the interaction between inhibitors and amyloid proteins and can potentially be a common tool for amyloid research. Strikingly, the SERS signal of COG corresponds very well with the state of protein fibrillation, hinting its function as an amyloid SERS signal amplifier. Therefore, this study provides a new means to monitor and interfere amyloid fibrillation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A class of novel δ-sulfonolactone-fused pyrazole scaffold was prepared via sulfur (VI) fluoride exchange (SuFEx) chemistry using aryl sulfonyl fluorides and pyrazolones. Enzyme screening revealed their cholinesterase inhibitory activity, among them, compounds 4a, 5a and 5d were identified as highly selective submicromolar BuChE inhibitors (IC 50  = 0.20, 0.46 and 0.42 μM, respectively), which exhibited nontoxicity, lipophilicity and remarkable neuroprotective activity. Kinetic studies showed that BuChE inhibition of compounds 5a and 5d was reversible, mixed-type and non-competitive inhibition against BuChE (K i  = 145 nM and 60 nM, respectively). Compound 5d can be accommodated into hBuChE via π-S interaction and hydrophobic interactions. The title compounds are potentially symptomatic treatment in progressive Alzheimer's disease., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Objective: Myocardial ischemia reperfusion is a major cause of cell injury during cardiac transplantation and is responsible for increased graft rejection. Several in vitro studies demonstrated the protective effect of P2Y11-like purinoreceptor stimulation in the context of myocardial ischemia/reperfusion. In this study, we hypothesized a possible cardioprotective role of P2Y11R stimulation against ischemia/reperfusion lesions and validated its clinical effect in vivo in a heart transplantation model., Methods: We subjected H9c2 rat cardiomyocyte-derived cell line to 5 hours of hypoxia and 1 hour of reoxygenation. P2Y11R selective agonist NF546 and antagonist NF340 were added at the onset of reoxygenation. Cell injuries were assessed by microculture tetrazolium reduction and intracellular adenosine triphosphate level. Clinical effect of P2Y11R stimulation was further investigated in vivo. Hearts from BALB/c mice were transplanted intra-abdominally into allogenic C57BL/6 mice (n = 104). Recipient mice were injected with P2Y11R agonist. Mice in the sham group were injected with saline solution. In the control group, hearts from C57BL/6 were transplanted into syngeneic C57BL/6 mice. Rejection lesions were investigated using histology and immunohistochemistry at days 3, 5, and 7 after transplantation. We measured caspase activities to quantify apoptosis. Production of proinflammatory and anti-inflammatory cytokines was investigated., Results: P2Y11R stimulation at the onset of reoxygenation significantly reduced in vitro hypoxia/reoxygenation injuries. This protection was suppressed with P2Y11R antagonist. In vivo, cardiac allograft survival was significantly prolonged after P2Y11R stimulation. Rejection lesions, classified according to the International Society of Heart Lung Transplantation guidelines and quantified using the mean number of inflammatory cells per field, were significantly reduced in the treated group. At day 5 after transplantation, P2Y11R agonist pretreated allografts also demonstrated less apoptotic lesions., Conclusions: Our data suggest a novel cardioprotective role of P2Y11R at the onset of reoxygenation/reperfusion against reperfusion injuries. Pharmacologic conditioning using P2Y11 agonist may be beneficial after cardiac transplantation in improving myocardial ischemia/reperfusion outcomes and decreasing graft rejection lesions., (Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Abextide, synthesized by conjugating an albumin-binding moiety-truncated Evans blue-to glucagon-like peptide 1 receptor (GLP-1R) agonist exendin-4, shows extended drug release and enhanced hypoglycemic effect in diabetic mice. The aim of this study is to evaluate the pharmacodynamics of Abextide in nonhuman primates. Two batches of elderly cynomolgus monkeys with naturally occurring diabetes are used for this study. During the whole experiment period, no abnormalities such as swelling at the injection site, lethargy, or hypoglycemia are observed in all animals. The monkeys in the Abextide group lose appetite after drug administration and then recover over time. In the single dose treatment, at day 1 and day 3 after treatment, decreased plasma glucose level is observed in the Abextide-treated group but not in placebo or Albiglutide-treated group. For monkeys that receive two doses of drug, the blood glucose level in all subjects in Abextide group decreases rapidly upon drug administration and return to a plateau by day 3. A similar pattern of response is seen after the second dose administration. The delayed drug release and hypoglycemic effect of Abextide make it potentially useful as an antidiabetic drug for weekly subcutaneous administration., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tissue remodeling of subepithelial mesenchymal cells is a major pathologic condition of chronic obstructive pulmonary disease and asthma. Fibroblasts contribute to fibrotic events and inflammation in both airway diseases. Recent mechanistic studies established a link between mitochondrial dysfunction or aberrant biogenesis leading to tissue remodeling of the airway wall in asthma. Protein arginine methyltransferase-1 (PRMT1) participated in airway wall remodeling in pulmonary inflammation. This study investigated the mechanism by which PRMT1 regulates mitochondrial mass in primary human airway wall fibroblasts. Fibroblasts from control or asthma patients were stimulated with TGF-β for up to 48 h, and the signaling pathways controlling PRMT1 expression and mitochondrial mass were analyzed. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI-1. The SMAD2/3 pathway was blocked by SB203580 and C/EBPβ by small interference RNA treatment. The data obtained from unstimulated cells showed a significantly higher basal expression of PRMT1 and mitochondrial markers in asthmatic compared with control fibroblasts. In all cells, TGF-β significantly increased the expression of PRMT1 through SMAD2/3 and C/EBPβ. Subsequently, PRMT1 upregulated the expression of the mitochondria regulators PGC-1α and heat shock protein 60. Both the inhibition of the SAMD2/3 pathway or PRMT1 attenuated TGF-β-induced mitochondrial mass and C/EBPβ and α-SMA expression. These findings suggest that the signaling sequence controlling mitochondria in primary human lung fibroblasts is as follows: TGF-β→SMAD2/3→C/EBPβ→PRMT1→PGC-1α. Therefore, PRMT1 and C/EBPβ present a novel therapeutic and diagnostic target for airway wall remodeling in chronic lung diseases., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Aim: To propose a new scoring system in the assessment of ocular surface epithelial damage in vernal keratoconjunctivitis (VKC)., Methods: 25 consecutive patients with VKC (50 eyes) were evaluated using the Quality of Life in children with VKC (QUICK) questionnaire and objective clinical measures: fluorescein and lissamine green staining and cornea confocal microscopy (Heidelberg Retina Tomography 3). Oxford, Van Bljsterweld and a new system, the VKC-Collaborative Longitudinal Evaluation of Keratoconus study (CLEK) (VKC-CLEK) scores, were used to evaluate the epithelial damage after staining., Results: Mean Oxford and VKC-CLEK scores were significantly different after fluorescein staining (P<0.001), but significantly correlated (P<0.001; r=0.649). The same data were obtained comparing Van Bljsterweld and VKC-CLEK after lissamine green staining (P<0.001; r=0.760). In patient with limbal VKC, a statistically significant difference was found comparing new VKC-CLEK scores and Oxford or Van Bljsterweld scores (P<0.001), but not in tarsal VKC. A statistically superior concordance was found between QUICK and VKC-CLEK scores compared with standard staining scores values (P<0.001)., Conclusions: Oxford and Van Bijsterveld scores are not adequate for the evaluation of the epithelial damage in patients with limbal VKC because the staining patterns considered for these tests do not correspond to the staining patterns in patients with VKC. We propose a new scoring system, VKC-CLEK, to better evaluate both limbal and tarsal epithelial damage in patients with VKC., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Adenosine triphosphate (ATP) is known to induce cell death in T lymphocytes at high extracellular concentrations. CD4 + and CD8 + T lymphocytes have a differential response to ATP, which in mice is due to differences in the P2X7 receptor expression levels. By contrast, we observed that the difference in human CD4 + and CD8 + T lymphocyte response toward the synthetic ATP-analog BzATP is not explained by a difference in human P2X7 receptor expression. Rather, the BzATP-induced human P2X7 receptor response in naïve and immune-activated lymphocyte subtypes correlated with the expression of another ATP-binding receptor: the human P2Y 11 receptor. In a recombinant expression system, the coexpression of the human P2Y 11 receptor counteracted BzATP-induced human P2X7 receptor-driven lactate dehydrogenase release (a marker of cell death) and pore formation independent of calcium signaling. A mutated non-signaling human P2Y 11 receptor had a similar human P2X7 receptor-inhibitory effect on pore formation, thus demonstrating that the human P2X7 receptor interference was not caused by human P2Y 11 receptor signaling. In conclusion, we demonstrate an important species difference in the ATP-mediated cell death between mice and human cells and show that in human T lymphocytes, the expression of the human P2Y 11 receptor correlates with human P2X7 receptor-driven cell death following BzATP stimulation.

Protein arginine methyltransferase 5 (PRMT5), a type II PRMT, is highly expressed in several types of tumors including cervical cancer. Arginine methyltransferase inhibitor 1 (AMI-1) inhibits solid tumors by targeting PRMT5. However, the effect of AMI-1 on cervical cancer is still unknown. In this study, we provided the first evidence that AMI-1 reduced cervical cancer cell proliferation, colony formation and promoted cell apoptosis in vitro. Suppression of tumorigenicity was also confirmed in vivo. Mechanistic studies revealed that AMI-1 significantly reduced PRMT5 level in cells and mice xenografts model of cervical cancer. These results suggest that AMI-1 inhibits cervical cancer by type II PRMT5.

Objective: To investigate the effect of semen-derived enhancer of virus infection (SEVI) on the infection of transmitted/founder (TF) HIV-1 and its matched chronic control (CC) viruses and the antagonism of ADS-J1 on SEVI-mediated enhancement of TF and CC virus infection in vitro., Methods: PAP 248-286 self-assembling into SEVI amyloid fibrils was validated by ThT assay. We generated the virus stocks of TF and CC virus pair. TZM-bl cells were infected with the mixture of SEVI and TF or CC viruses for 72 h. Luciferase activity was used to observe the enhancement of SEVI. SEVI was treated with different concentrations of ADS-J1 and incubated with TF or CC viruses. TZM-bl cells were then infected with the mixture and luciferase activity was detected 72 h after infection to analyze the antagonism of ADS-J1 on the enhancing effect of SEVI. ADS-J1 was also incubated with TF and CC viruses directly and TZM-bl cells were infected for 72 h to evaluate the antiviral effect using luciferase assay. SEVI was treated with ADS-J1 and Zeta potential was determined to explore the antagonistic mechanism of ADS-J1., Results: ThT assay showed that PAP 248-286 was capable of self-assembly into SEVI amyloid fibrils. SEVI significantly accelerated TF and CC viruses infection (P<0.05), and ADS-J1 not only significantly antagonized the enhancement of SEVI (P<0.05) but also directly inhibited the infection of TF and CC viruses (P<0.05). ADS-J1 neutralized the positive charge of SEVI in a dose-dependent manner., Conclusions: SEVI promotes the infection of TF and CC strains, and ADS-J1 antagonizes SEVI-mediated enhancement of TF and CC viruses by neutralizing the positive charge of SEVI.

Mycobacterium tuberculosis (MTB) is the causative agent of pulmonary tuberculosis. Rapid and accurate diagnosis is crucial to tuberculosis control and prevention. A series of diagnostic methods has been available for MTB detection; however, new rapid, simple and affordable methods are needed. In this study, a multiple cross displacement amplification (MCDA)-based assay was developed to detect the IS6110 gene of the M. tuberculosis complex. Hydroxy naphthol blue (HNB), a colorimetric indicator, was used to detect amplification products. Amplification was carried out at a constant temperature (68°C) for only 40min, followed by direct determination of amplification products through observation of color variations. The entire detection procedure, from processing of specimens to reading of results, required only 85min. Moreover, this assay, hereafter designated MTB-MCDA-HNB, was able to detect as little as 1pg of DNA extracted from the Bacille Calmette-Guerin (BCG) strain of Mycobacterium bovis. No cross-reaction with nontuberculous mycobacteria (NTM) species was observed. Moreover, during testing of clinical samples, the sensitivity and specificity of MCDA results were 94.7% and 92.9%, respectively, when compared to results obtained using the Xpert MTB/RIF method. Therefore, the MTB-MCDA-HNB method developed in this study holds promise for application as an effective point-of-care test to detect M. tuberculosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The chemical amaranth (AM) is permitted as a colouring agent in a variety of foods. Safety was established based on chronic rodent studies. AM and its metabolite naphthionic acid (NA) can be absorbed through the intestine, exposing circulating immune cells including splenocytes. An AM feeding study in rats demonstrated an increase in blood lymphocytes. Yet, in contrast, AM inhibited the delayed-type hypersensitivity reaction to antigen. DO11.10 mice express a T Cell Receptor specific for ovalbumin 323-339 peptide (OVAp) presented by I-A d MHCII. DO11.10 splenocytes were cultured to evaluate mechanisms by which AM and NA modulate immune cell function in vitro. Exposure to OVAp alone for 72 h induced cell proliferation, and combination with 2 or 20 μg/mL AM increased IFN-γ. Cytotoxicity was evident at higher concentrations of AM (200 and 2000 μg/mL) and NA (2000 μg/mL) in combination with OVAp, as both cell number and cytokine secretion decreased. At 200 μg/mL AM with OVAp, immunotoxicity gene expression was modified and OVAp-specific KJ1-26 + CD28 + cells became enriched. The equivalent dose of NA did not modify those parameters. Using an antigen-specific model in vitro, lower concentrations of AM potentiated pro-inflammatory cytokine production, and higher concentrations of AM and NA demonstrated cytotoxicity., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Letermovir, GW275175X (a benzimidazole), and tomeglovir (Bay38-4766) are chemically unrelated human cytomegalovirus (CMV) terminase complex inhibitors that have been tested in human subjects. UL56 gene mutations are the dominant pathway of letermovir resistance, while UL89 and UL56 mutations are known to confer benzimidazole resistance. This study compares the mutations elicited by the three inhibitors during in vitro CMV propagation. GW275175X consistently selected for UL89 D344E and sometimes selected for UL89 C347S, UL89 R351H, or UL56 Q204R. Tomeglovir consistently selected for UL89 V362M and sometimes selected for UL89 N329S, T350M, H389N, or N405D or UL56 L208M, E407D, H637Q, or V639M. Adding to known and novel UL56 mutations, letermovir occasionally selected for UL89 N320H, D344E, or M359I. Recombinant phenotyping confirmed that UL89 D344E conferred 9-fold resistance (an increased 50% effective concentration [EC 50 ]) for GW275175X and increased the letermovir and tomeglovir EC 50 s by 1.7- to 2.1-fold for the baseline virus and the UL56 Q204R, E237D, F261L, and M329T mutants. UL89 N320H and M359I conferred <2-fold letermovir resistance but 7-fold tomeglovir resistance; the N320H mutant was also 4-fold resistant to GW275175X. UL89 N329S conferred tomeglovir and letermovir cross-resistance. UL89 T350M conferred resistance to all three inhibitors. UL89 C347S conferred 27-fold GW275175X resistance. UL89 V362M and H389N conferred 98-fold and 29-fold tomeglovir resistance, respectively, without conferring cross-resistance. Thus, characteristic UL89 mutations confer substantial resistance to GW275175X and tomeglovir and are an uncommon accessory pathway of letermovir resistance. Instances of moderate cross-resistance and the proximity of the selected UL89 and UL56 mutations suggest targeting of a similar terminase functional locus involving UL56 and UL89 interaction., (Copyright © 2017 American Society for Microbiology.)

There is a growing body of evidence pointing to the role of purinergic signaling in the development and progression of various conditions that have inflammation as a common pathogenetic denominator. The aim of the present study was to assess the involvement of P 2 Y 11 purinergic receptors in the regulation of vascular function in aortic segments obtained using an experimental model of acute inflammation, the lipopolysaccharide (LPS, 8 mg/kg, i.p)-treated rats. Twelve hours after LPS administration, thoracic aortas were isolated and used for studies of vascular reactivity in the organ bath and for the measurement of reactive oxygen species (ROS) generation, respectively. LPS treatment significantly increased contractility to phenylephrine and attenuated the endothelium-dependent relaxation of the vascular segments in response to acetylcholine; an increased production of hydrogen peroxide (H 2 O 2 ) was also recorded. The P 2 Y 11 activator, NF 546, decreased the LPS-induced aortic H 2 O 2 release and partially normalized the vasomotor function, namely reduced contractility and improved relaxation. The effect was abolished by co-treatment with the P 2 Y 11 inhibitor, NF 340, and also after endothelium denudation. Importantly, NF 546 did not elicit an antioxidant effect by acting as a H 2 O 2 scavenger, suggesting that the beneficial outcome of this treatment on the vasculature is the consequence of P 2 Y 11 stimulation. In conclusion, purinergic P 2 Y 11 receptors stimulation improves vascular function and mitigates oxidative stress in the setting of acute systemic inflammation, revealing salutary effects and therapeutic potential in pathologies associated with endothelial dysfunction.

Extracellular ATP-induced Ca2+ signalling is critical in regulating diverse physiological and disease processes. Emerging evidence suggests high concentrations of extracellular ATP in tumour tissues. In this study, we examined the P2 receptor for ATP-induced Ca2+ signalling in human hepatocellular carcinoma (HCC) cells. Fura-2-based measurements of the intracellular Ca2+ concentration ([Ca2+]i) showed that extracellular ATP induced an increase in the [Ca2+]i in human HCC Huh-7 and HepG2 cells. NF546, a P2Y11 receptor agonist was equally effective in inducing an increase in the [Ca2+]i. In contrast, agonists for the P2X receptors (αβmeATP and BzATP), P2Y1 receptor (MRS2365) or P2Y2 receptor (MRS2768) were ineffective. In addition, ATP/NF546-induced increases in the [Ca2+]i were strongly inhibited by treatment with NF340, a P2Y11 receptor antagonist. Immunofluorescent confocal imaging and western blotting analysis consistently demonstrated the P2Y11 receptor expression in Huh-7 and HepG2 cells. Transfection with P2Y11-specific siRNA attenuated the P2Y11 receptor protein expression level and also reduced NF546-induced increase in the [Ca2+]i. Importantly, immunohistochemistry revealed that the P2Y11 receptor was expressed at very high level in human HCC tissues and, by contrast, it was barely detected in normal liver tissues. Trans-well cell migration assay demonstrated that ATP and NF546 induced concentration-dependent stimulation of Huh-7 cell migration. Treatment with NF340 prevented ATP-induced stimulation of cell migration. Taken together, our results show carcinoma-specific expression of the P2Y11 receptor and its critical role in mediating ATP-inducing Ca2+ signalling and regulating cell migration in human HCC cells.

This letter describes the discovery, synthesis, SAR, and biological activity of [2.2.1]-bicyclic sultams as potent antagonists of the androgen receptor. Optimization of the series led to the identification of compound 25, which displayed robust pharmacodynamic effects in rats after oral dosing., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Posttranslational modifications of histone tails can alter chromatin structure and regulate gene transcription. While recent studies implicate the lysine/arginine protein methyltransferases in the regulation of genes for endothelial metabolism, the role of AMI-1 and AMI-5 compounds in angiogenesis remains unknown. Here, we show that global inhibition of arginine and lysine histone methyltransferases (HMTs) by AMI-5 induced an angiostatic profile in human microvascular endothelial cells and human umbilical vein endothelial cells. Based on FACS analysis, we found that inhibition of HMTs significantly affects proliferation of endothelial cells, by suppressing cell cycle progression in the G 0 /G 1 phase. Immunofluorescent studies of the endothelial cells replication pattern by 5-ethynyl-2'-deoxyuridine incorporation disclosed that AMI-5, and the arginine methyltransferase inhibitor AMI-1, induced heterochromatin formation and a number of nuclear abnormalities, such as formation of micronuclei (MNs) and nucleoplasmic bridges (NPBs), which are markers of chromosomal instability. In addition to the modification of the cell cycle machinery in response to AMIs treatment, also endothelial cells migration and capillary-like tube formation processes were significantly inhibited, implicating a stimulatory role of HMTs in angiogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

It has been reported that some of the food additives may cause sensitization, inflammation of tissues, and potentially risk factors in the development of several chronic diseases. Thus, we hypothesized that expressions of common inflammatory molecules - known to be involved in the development of various inflammatory conditions and cancers - are affected by these food additives. We investigated the effects of commonly used food preservatives and artificial food colorants based on the expressions of NFκB, GADD45α, and MAPK8 (JNK1) from the tissues of liver. RNA was isolated based on Trizol protocol and the activation levels were compared between the treated and the control groups. Tartrazine alone could elicit effects on the expressions of NFκB (p = 0.013) and MAPK8 (p = 0.022). Azorubine also resulted in apoptosis according to MAPK8 expression (p = 0.009). Preservatives were anti-apoptotic in high dose. Sodium benzoate (from low to high doses) dose-dependently silenced MAPK8 expression (p = 0.004 to p = 0.002). Addition of the two preservatives together elicited significantly greater expression of MAPK8 at half-fold dose (p = 0.002) and at fivefold dose (p = 0.008). This study suggests that some of the food preservatives and colorants can contribute to the activation of inflammatory pathways.

In the treatment of type 2 diabetes mellitus, it is very important to develop therapeutics with prolonged circulation half-life. Exendin-4 is a glucagon like peptide-1 receptor (GLP-1R) agonist that has been modified in different ways for imaging insulinoma and for treating type-2 diabetes. In this work, we synthesized a maleimide derivative of truncated Evans blue dye (MEB-C3-Mal) to conjugate with (Cys(40))exendin-4 to obtain a highly stable MEB-C3-(Cys(40))exendin-4 (denoted as Abextide II). Through in situ binding with endogenous albumin, Abextide II lowers blood glucose level and prolongs the hypoglycemic effect in a type 2 diabetes mouse model more than the FDA approved Albiglutide., Competing Interests: The authors declare no competing financial interest.

In view of the emerging clinical indications for Phosphodiesterase 9 inhibitors e.g. treatment of Alzheimer, diabetes, cancer, and the limited number of its selective inhibitors which possess a single chemical scaffolds, a structure-based approach was undertaken to mine the ZINC database by virtual screening to identify novel PDE9 inhibitors. The database, which was never reported to have been used before for discovery of PDE9 inhibitors, was screened against the ligand binding pocket of the PDE9 complex (PDB:4GH6) using molecular docking programs, MOE and AutoDock Vina in PyRx. Three different scoring functions were used to evaluate the docking poses and scores of the compounds, and the compounds were selected through consensus selection, thus reducing the margin of error in docking. The highest scoring compounds were then selected and purchased for in vitro testing as PDE9 inhibitors and cancer growth inhibitory agents. This led to the discovery of three previously unreported potent PDE 9 inhibitory compounds with two unique chemical scaffolds. Consistent with the role of PDE9 in cancer cell growth, the compounds also inhibited the growth of breast tumor cell lines, MCF-7 and MDA-468 at concentrations comparable to those that inhibited PDE9.

Background: Protein arginine methyltransferase 5 (PRMT5), a type II PRMT, is highly expressed in some tumors, but its role in hepatocellular carcinoma (HCC) is still unknown., Methods: PRMT5 level in HCC specimens was determined by immunohistochemical staining and the association with clinicopathologic features was evaluated. PRMT5 was inhibited by AMI-1 (a small molecule inhibitor of PRMTs) or small interference RNA (siRNA). The proliferation of HCC cells was tested by Cell Counting Kit-8, cell migration was evaluated by Transwell assay and cell cycle and apoptosis were analyzed by flow cytometry. The effect of AMI-1 on HCC in vivo was examined by mouse xenograft model., Results: PRMT5 expression was markedly upregulated in HCC tissues, and correlated inversely with overall patient survival. Knockdown of PRMT5 significantly reduced the proliferation of HCC cells, but did not affect the growth of normal liver cells. Furthermore, β-catenin was identified as a target of PRMT5. Silencing PRMT5 significantly down-regulated the expression of β-catenin and the downstream effector Cyclin D1 in HCC cells. AMI-1 strongly inhibited HCC growth in vivo, increased the ratio of Bax/Bcl-2, and led to apoptosis and loss of migratory activity in several HCC cells. Meanwhile, AMI-1 decreased the expression levels of symmetric dimethylation of H4 (H4R3me2s), a histone mark of PRMT5., Conclusions: PRMT5 plays an important role in HCC. PRMT5 may be a promising target for HCC therapy.

Protein arginine methyltransferases (PRMTs) plays critical roles in cancer. PRMT5 has been implicated in several types of tumors. However, the role of PRMT5 in cancer development remains to be fully elucidated. Here, we provide evidence that PRMT5 is overexpressed in colorectal cancer (CRC) cells and patient-derived primary tumors, correlated with increased cell growth and decreased overall patient survival. Arginine methyltransferase inhibitor 1 (AMI-1)strongly inhibited tumor growth, increased the ratio of Bax/Bcl-2, and induced apoptosis in mouse CRC xenograt model. AMI-1 also induced apoptosis and decreased the migratory activity in several CRC cells. In CRC xenografts AMI-1 significantly decreased symmetric dimethylation of histone 4 (H4R3me2s), a histone mark of type II PRMT5, but not the expression of H4R3me2a, a histone mark of type I PRMTs. These results suggest that the inhibition of PRMT5 contributes to the antitumor efficacy of AMI-1. Chromatin immunoprecipitation (ChIP) identified FGFR3 and eIF4E as two key genes regulated by PRMT5. PRMT5 knockdown reduced the levels of H4R3me2s and H3R8me2s methylation on FGFR3 and eIF4E promoters, leading to decreased expressions of FGFR3 and eIF4E. Collectively, our findings provide new evidence that PRMT5 plays an important role in CRC pathogenesis through epigenetically regulating arginine methylation of oncogenes such as eIF4E and FGFR3.

Protein arginine methyltransferase (PRMT)1, methylating both histones and key cellular proteins, has emerged as a key regulator of various cellular processes. This study aimed to identify the mechanism that regulates PRMT1 in chronic Ag-induced pulmonary inflammation (AIPI) in the E3 rat asthma model. E3 rats were challenged with OVA for 1 or 8 wk to induce acute or chronic AIPI. Expression of mRNAs was detected by real-time quantitative PCR. PRMT1, TGF-β, COX2, and vascular endothelial growth factor protein expression in lung tissues was determined by immunohistochemistry staining and Western blotting. In the in vitro study, IL-4-stimulated lung epithelial cell (A549) medium (ISEM) with or without anti-TGF-β Ab was applied to human fibroblasts from lung (HFL1). The proliferation of HFL1 was determined by MTT. AMI-1 (pan-PRMT inhibitor) was administered intranasally to chronic AIPI rats to determine PRMT effects on asthmatic parameters. In lung tissue sections, PRMT1 expression was significantly upregulated, mainly in epithelial cells, in acute AIPI lungs, whereas it was significantly upregulated mainly in fibroblasts in chronic AIPI lungs. The in vitro study revealed that ISEM elevates PRMT1, COX2, and vascular endothelial growth factor expressions, and it promoted fibroblast proliferation. The application of anti-TGF-β Ab suppressed COX2 upregulation by ISEM. AMI-1 inhibited the expression of COX2 in TGF-β-stimulated cells. In the in vivo experiment, AMI-1 administered to AIPI rats reduced COX2 production and humoral immune response, and it abrogated mucus secretion and collagen generation. These findings suggested that TGF-β-induced PRMT1 expression participates in fibroblast proliferation and chronic airway inflammation in AIPI., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Angiogenesis, the process of neovascularization, plays an important role in physiological and pathological conditions. ST104P is a soluble polysulfated-cyclo-tetrachromotropylene compound with anti-viral and anti-thrombotic activities. However, the functions of ST104P in angiogenesis have never been explored. In this study, we investigated the effects of ST104P in angiogenesis in vitro and in vivo. Application of ST104P potently suppressed the microvessels sprouting in aortic rings ex vivo. Furthermore, ST104P treatment significantly disrupted the vessels' development in transgenic zebrafish in vivo. Above all, repeated administration of ST104P resulted in delayed tumor growth and prolonged the life span of mice bearing Lewis lung carcinoma. Mechanistic studies revealed that ST104P potently inhibited the migration, tube formation and wound closure of human umbilical endothelial cells (HUVECs). Moreover, ST104P treatment inhibited the secretion and expression of matrix metalloproteinase-2 (MMP-2) in a dose-dependent manner. Together, these results suggest that ST104P is a potent angiogenesis inhibitor and may hold potential for treatment of diseases due to excessive angiogenesis including cancer.

The aberrant activation of STAT3 occurs in many human cancers and promotes tumor progression. Phosphorylation of a tyrosine at amino acid Y705 is essential for the function of STAT3. Synthesized carbazole derived with fluorophore compound 12 was discovered to target STAT3 phosphorylation. Compound 12 was found to inhibit STAT3-mediated transcription as well as to reduce IL-6 induced STAT3 phosphorylation in cancer cell lines expressing both elevated and low levels of phospho-STAT3 (Y705). Compound 12 potently induced apoptosis in a broad number of TNBC cancer cell lines in vitro and was effective at inhibiting the in vivo growth of human TNBC xenograft tumors (SUM149) without any observed toxicity. Compound 12 also effectively inhibited the growth of human lung tumor xenografts (A549) harboring aberrantly active STAT3. In vitro and in vivo studies showed that the inhibitory effects of 12 on phospho-STAT3 were through up-regulation of the protein-tyrosine phosphatase PTPN6. Our present studies strongly support the continued preclinical evaluation of compound 12 as a potential chemotherapeutic agent for TNBC and cancers with constitutive STAT3 signaling.

The enzymatic activities of protein methyltransferases serve to write covalent modifications on histone and non-histone proteins in the control of gene transcription. Here, we describe gene expression changes in human endothelial cells caused by treatment with methyltransferase inhibitors 7,7'-carbonylbis (azanediyl) bis(4-hydroxynaphthalene-2 -sulfonic acid (AMI-1) and disodium-2-(2,4,5,7- tetrabromo-3-oxido-6-oxoxanthen-9-yl) benzoate trihydrate (AMI-5). Deep sequencing of mRNA indicated robust change on transcription following AMI-5 treatment compared with AMI-1. Functional annotation analysis revealed that both compounds suppress the expression of genes associated with translational regulation, suggesting arginine methylation by protein arginine methyltransferases (PRMTs) could be associated with regulation of this pathway. Interestingly, AMI-5 but not AMI-1 was found to decrease methylation of H3 histones at lysine 4 and down-regulate gene expression associated with interleukin-6 (IL-6) and activator protein-1 (AP-1) signaling pathways. These results imply that inhibition of protein methylation by AMI-1 and AMI-5 can differentially regulate specific pathways with potential to interrupt pathological signaling in the vascular endothelium., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ultrafiltration membrane processes have become an established technology in the treatment and reuse of secondary effluents. Nevertheless, membrane fouling arises as a major obstacle in the efficient operation of these systems. In the current study, the performance of tubular ultrafiltration ceramic membranes was evaluated according to the roles exerted by membrane pore size, transmembrane pressure and feed concentration on a binary foulant system simulating textile wastewater. For that purpose, carboxymethyl cellulose sodium salt (CMC) and an azo dye were used as colloidal and organic foulants, respectively. Results showed that a larger pore size enabled more solutes to get adsorbed into the pores, producing a sharp permeate flux decline attributed to the rapid pore blockage. Besides, an increase in CMC concentration enhanced severe fouling in the case of the tighter membrane. Concerning separation efficiency, organic matter was almost completely removed with removal efficiency above 98.5%. Regarding the dye, 93% of rejection was achieved. Comparable removal efficiencies were attributed to the dynamic membrane formed by the cake layer, which governed process performance in terms of rejection and selectivity. As a result, none of the evaluated parameters showed significant influence on separation efficiency, supporting the significant role of cake layer on filtration process., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We previously identified a potent small-molecule human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, termed ADS-J1, and hypothesized that it mainly targeted the hydrophobic pocket in the gp41 N-terminal heptad repeat (NHR) trimer. However, this hypothesis has been challenged by the fact that ADS-J1 cannot induce drug-resistance mutation in the gp41 pocket region. Therefore, we show herein that HIV-1 mutants resistant to T2635, a peptide derived from the gp41 C-terminal heptad repeat (CHR) region with pocket-binding domain (PBD), were also resistant to ADS-J1. We also show that pseudoviruses with mutations at positions 64 and 67 in the gp41 pocket region were highly resistant to ADS-J1 and C34, another CHR-peptide with PBD, but relatively sensitive to T20, a CHR-peptide without PBD. ADS-J1 could effectively bind to N36Fd, a mimic of the gp41 NHR-trimer with pocket exposed, and block binding of C34 to N36Fd trimer to form six-helix bundle (6-HB). However, ADS-J1 was less effective in binding to N36Fd trimer with mutations in the gp41 pocket region, such as N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd, and N36(Q66R)Fd, as well as less effective in blocking 6-HB formation between C34 and these mutant N36Fd trimers. These results confirm that ADS-J1 mainly targets the pocket region in the HIV-1 gp41 NHR trimer and suggest that it could be used as a lead for developing small-molecule HIV fusion inhibitors and as a molecule probe for studying the mechanisms of gp41-mediated membrane fusion., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Noroviruses are members of the Caliciviridae family of positive sense RNA viruses. In humans Noroviruses cause rapid onset diarrhea and vomiting. Currently Norovirus infection is responsible for 21 million gastroenteritis yearly cases in the USA. Nevertheless, despite the obvious public health and socio-economic relevance, no effective vaccines/antivirals are yet available to treat Norovirus infection. Since the activity of RNA-dependent RNA polymerase (RdRp) plays a key role in genome replication and in the synthesis/amplification of subgenomic RNA, the enzyme is considered a promising target for antiviral drug development. In this context, following the identification of suramin and NF023 as Norovirus RdRp inhibitors, we analyzed the potential inhibitory role of naphthalene di-sulfonate (NAF2), a fragment derived from these two molecules. Although NAF2, tested in enzymatic polymerase inhibition assays, displayed low activity against RdRp (IC50=14μM), the crystal structure of human Norovirus RdRp revealed a thumb domain NAF2 binding site that differs from that characterized for NF023/suramin. To further map the new potential inhibitory site, we focused on the structurally related molecule pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) tetrasodium salt (PPNDS). PPNDS displayed below-micromolar inhibitory activity versus human Norovirus RdRp (IC50=0.45μM), similarly to suramin and NF023. Inspection of the crystal structure of the RdRp/PPNDS complex showed that the inhibitor bound to the NAF2 thumb domain site, highlighting the relevance of such new binding site for exploiting Norovirus RdRp inhibitors., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The aim of the study was to identify a signalling pathway allowing NAADP-induced intracellular NAADP increase and involving the P2Y11-like receptor. P2Y11-like and β-adrenergic receptors may play important regulatory roles within the cardiovascular system. Both receptors have been shown to be involved in triggering myocardial preconditioning. Using a Langendorff model we report a positive inotropic response induced by extracellular NAADP via P2Y11-like receptor stimulation. In cardiomyocyte cultures, P2Y11-like receptor stimulation by extracellular NAADP ([NAADP]e) increased intracellular cADP-ribose and NAADP concentration as evidenced by direct measurements. NF546, a new selective P2Y11 receptor agonist, increased intracellular cAMP, cADP-ribose and NAADP concentration confirming the involvement of the P2Y11-like receptor in this signalling pathway. NF157, a P2Y11 receptor antagonist, suppressed the increase in intracellular cADPr, NAADP and NAAD induced by either [NAADP]e or NF546. The response profile for intracellular cADP-ribose and NAADP concentration following P2Y11-like stimulation with NF546 was similar to reported data relating β-adrenergic stimulation with isoprenaline. This response represents the signature of the Gs/ADP-ribosyl cyclase activity. Moreover, this study provides a signalling pathway: intracellular NAADP increase induced by extracellular NAADP via metabotropic activity of P2Y11-like receptor. This pathway implying P2Y11-like could take part in the intracellular calcium rise reported for extracellular NAADP., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The effect of the auxin physiological analogues analogues 1-[2-chloroethoxycarbonylmethyl]-4-naphthalenesulfonic acid calcium salt (TA-12) and 1-[2-dimethylaminoethoxicarbonylmethyl]naphthalene chlormethylate (TA-14) TA-14 on different winter rapeseed cultivars were studied with regard to their autumnal growth, cold hardening, accumulation of the stress-protective metabolites proline and saccharide in plant organs: apical bud and root collum, winter survival and productivity formation. The test cultivars were the very early 'Libea' medium-resistant to wintering, the medium-early 'Sunday' resistant to wintering, the medium-early 'Valesca' less than medium resistant to wintering, and the early 'Hornet' (hybrid) tolerant to stress growth conditions. During the period of cold hardening in natural field conditions, the test compounds TA-12 (2 mM) and TA-14 (4 mM), applied to different winter rapeseed cultivars at the 4th-5th leaf stage, stimulate accumulation proline and saccharides (sucrose and glucose) in the root collum and apical bud tissues, influence plants acclimation to cold, overwintering and productivity formation. Compounds TA-12 and especially TA-14 produced a stable effect on seed and crude fat yield in cvs. 'Hornet', 'Sunday' and 'Libea'. The genotypic peculiarities of a cultivar and the meteorological conditions of the plant vegetation period were the factors that mostly determined fatty acid content in seed oil.

Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploiting in vitro priming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 μM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives.

Silver nanoparticles (AgNPs) are increasingly used in daily life for their antibacterial properties, but their low stability and high cytotoxicity hamper practical applications. In this work, sodium 1-naphthalenesulfonate-functionalized reduced graphene oxide (NA-rGO) was used as a substrate for AgNPs to produce a AgNP-NA-rGO hybrid. This hybrid showed substantially higher antibacterial activity than polyvinyl pyrrolidone (PVP)-stabilized AgNPs, and the AgNPs on NA-rGO were more stable than the AgNPs on PVP, resulting in long-term antibacterial effects. More importantly, this hybrid showed excellent water solubility and low cytotoxicity, suggesting the great potential application as sprayable reduced graphene oxide based antibacterial solutions., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein arginine methyltransferases (PRMTs), catalyzing methylation of both histones and other cellular proteins, have emerged as key regulators of various cellular processes. This study aimed to identify key PRMTs involved in Ag-induced pulmonary inflammation (AIPI), a rat model for asthma, and to explore the role of PRMT1 in the IL-4-induced eosinophil infiltration process. E3 rats were i.p. sensitized with OVA/alum and intranasally challenged with OVA to induce AIPI. The expressions of PRMT1-6, eotaxin-1, and CCR3 in lungs were screened by real-time quantitative PCR. Arginine methyltransferase inhibitor 1 (AMI-1, a pan-PRMT inhibitor) and small interfering RNA-PRMT1 were used to interrupt the function of PRMT1 in A549 cells. In addition, AMI-1 was administrated intranasally to AIPI rats to observe the effects on inflammatory parameters. The results showed that PRMT1 expression was mainly expressed in bronchus and alveolus epithelium and significantly upregulated in lungs from AIPI rats. The inhibition of PRMTs by AMI-1 and the knockdown of PRMT1 expression were able to downregulate the expressions of eotaxin-1 and CCR3 with the IL-4 stimulation in the epithelial cells. Furthermore, AMI-1 administration to AIPI rats can also ameliorate pulmonary inflammation, reduce IL-4 production and humoral immune response, and abrogate eosinophil infiltration into the lungs. In summary, PRMT1 expression is upregulated in AIPI rat lungs and can be stimulated by IL-4. Intervention of PRMT1 activity can abrogate IL-4-dependent eotaxin-1 production to influence the pulmonary inflammation with eosinophil infiltration. The findings may provide experimental evidence that PRMT1 plays an important role in asthma pathogenesis.

Fibroblast growth factors (FGFs) are recognized targets for the development of therapies against angiogenesis-driven diseases, including cancer. The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity. Here by using a combination of techniques including Nuclear Magnetic Resonance, Molecular Dynamics, Surface Plasmon Resonance and cell-based binding assays we clarify the molecular mechanism of inhibition of an angiostatic small molecule, sm27, mimicking the endogenous inhibitor of angiogenesis, thrombospondin-1. NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions. The functional consequence of the inhibitor binding is an impaired FGF2 interaction with both its receptors, as demonstrated by SPR and cell-based binding assays. We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

Protein arginylation and arginine methylation are two posttranslational modifications of emerging importance that involve Arg residues and their modifications. To test a hypothesis that posttranslationally added arginines can be methylated, we used high-precision mass spectrometry and metabolic labeling to find whether posttranslationally added arginines can serve as methylation sites. We identified a number of proteins in vivo, on which posttranslationally added Arg have undergone mono- and dimethylation. This double modification predominantly affects the chromatin-containing nuclear fraction and likely plays an important regulatory role in chromatin-associated proteins. Moreover, inhibition of arginylation and Arg methylation results in a significant reduction of the nucleus size in cultured cells, suggesting changes in chromatin compaction and nuclear architecture. Our findings suggest a functional link between protein regulation by arginylation and methylation that affects nuclear structure in vivo., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Prophylactic vaccination against HIV-1 sexual transmission will probably require antibody elicitation at genital mucosal surfaces. However, HIV-1 envelope glycoprotein (Env)-based antigens are weakly immunogenic, particularly when applied mucosally. The polyanion PRO 2000 is safe for human vaginal application, and thus may represent a potential formulating agent for vaginal delivery of experimental vaccine immunogens. Based upon its biochemical properties, we hypothesized that PRO 2000 might enhance mucosal immunogenicity of HIV-1 envelope glycoprotein (Env)-based antigens, promoting local and systemic immune responses. Vaginal immunization with Env-PRO 2000 resulted in significantly increased titres of Env-specific mucosal IgA and IgG in mice and rabbits, respectively, compared to Env alone, revealing modest but significant mucosal adjuvant activity for PRO 2000. In vitro, PRO 2000 associated with Env, protecting the glycoprotein from proteolytic degradation in human vaginal lavage. Unexpectedly, PRO 2000 antagonized TLR4 activation, suppressing local production of inflammatory cytokines. Since inflammation-mediated recruitment of viral target cells is a major risk factor in HIV-1 transmission, the immune modulatory and anti-inflammatory activities of PRO 2000 combined with its intravaginal safety profile suggests promise as an HIV-1 mucosal vaccine formulating agent.

Defensins are highly abundant antimicrobial peptides in the female genital mucosa. We have previously shown that human defensins 5 and 6 (HD5 and HD6), produced by cervicovaginal epithelial cells, significantly enhance HIV infectivity in vitro. Candidate polyanion microbicides, including PRO 2000, cellulose sulfate and carrageenan, failed to protect women against HIV infection in large-scale clinical trials, but the molecular basis of ineffectiveness was not clear. We hypothesized that mucosal host factors such as HD5 an HD6 may alter the activity of polyanion microbicides against HIV. Our results demonstrated that HD5 and HD6 but not their linear analogs antagonized the anti-HIV activity of PRO 2000, cellulose sulfate and carrageenan in vitro. Polyanion microbicides also reduced the HIV-enhancing effect of these defensins. We conclude that mucosal host factors could negatively impact the efficacy of topical microbicides against HIV, and their impact on the activity of candidate microbicides needs to be considered during the preclinical evaluation., (Copyright © 2010 S. Karger AG, Basel.)

The use of microbicides is a promising approach for the prevention of HIV-1 transmission. Unfortunately, various candidates failed in clinical trials. In some cases, the candidate microbicide even resulted in enhanced virus transmission. Therefore, there is an urgent need to develop more predictive preclinical strategies to anticipate the in vivo efficiency/toxicity rate, including in vitro assays that evaluate effects on epithelial integrity and inflammation. The present study aims to identify potential safety issues concerning the use of microbicides and excipients commonly used in vaginal microbicide preparations. The toxicities of various active pharmaceutical ingredients (APIs; TMC-120, UC-781, tenofovir [PMPA], PRO-2000, and glycerol monolaurate [GML]) and excipients (preservatives, cosolvents, surfactants, and cyclodextrins) were evaluated using an in vitro dual-chamber model and uterine cervical explants. Epithelial viability and permeation of fluorescent virus-sized beads, as well as induction of interleukin-8 (IL-8; as a sensitive marker of an inflammatory response), were assessed. Surprisingly, cell viability and epithelial layer integrity were compromised by most excipients at concentrations near the typical concentration used in vaginal gels, and a significant increase in the production of IL-8 was observed at subtoxic concentrations. Within the APIs, TMC-120, UC-781, and PMPA showed higher selectivity indices than PRO-2000 and GML. In conclusion, identification of safety issues concerning the use of pharmaceutical excipients could help to formulate less toxic vaginal microbicide preparations.

We had shown that virus resistance to ADS-J1 was associated with amino acid changes in the envelope glycoprotein, mostly located in the gp120 coding region. Time-of-addition and endocytic virus transfer assays clearly demonstrated that ADS-J1 behaved as a gp120 inhibitor. ADS-J1-resistant virus was cross-resistant to the polyanion dextran sulfate, and recombination of gp120 recovered only the ADS-J1-resistant phenotype. In summary, ADS-J1 blocks an early step of virus entry that appears to be driven by gp120 alone.

Here we report the synthesis of a number of compounds structurally related to arginine methyltransferase inhibitor 1 (AMI-1). The structural alterations that we made included: 1) the substitution of the sulfonic groups with the bioisosteric carboxylic groups; 2) the replacement of the ureidic function with a bis-amidic moiety; 3) the introduction of a N-containing basic moiety; and 4) the positional isomerization of the aminohydroxynaphthoic moiety. We have assessed the biological activity of these compounds against a panel of arginine methyltransferases (fungal RmtA, hPRMT1, hCARM1, hPRMT3, hPRMT6) and a lysine methyltransferase (SET7/9) using histone and nonhistone proteins as substrates. Molecular modeling studies for a deep binding-mode analysis of test compounds were also performed. The bis-carboxylic acid derivatives 1 b and 7 b emerged as the most effective PRMT inhibitors, both in vitro and in vivo, being comparable or even better than the reference compound (AMI-1) and practically inactive against the lysine methyltransferase SET7/9.

The methylation of proteins is an important post-translational mechanism that has been established to influence the activity of nuclear and nucleic acid binding proteins. Much less is known about the importance of protein methylation in the regulation of cytosolic proteins. Increased methylation of proteins is observed in cardiovascular disease and occurs in conjunction with elevated production of reactive oxygen species. However, the nature of the relationship between reactive oxygen species and protein methylation is poorly understood. Therefore, the goal of the current study was to determine whether protein methylation influences the catalytic activity of the NADPH oxidases (Nox), which are a family of enzymes responsible for the generation of superoxide. We found that the selective inhibitor of protein arginine methyltransferases 7,7'-carbonylbis(azanediyl)bis(4-hydroxynaphthalene-2-sulfonic acid (AMI-1) was a potent antagonist of Nox-derived superoxide production. However, structurally and mechanistically dissimilar inhibitors of protein methylation and coexpression of protein arginine methyltransferase 1 did not influence Nox activity. Rather, the effect of AMI-1 was rapidly reversible and could be demonstrated in an assay using chemically synthesized superoxide. We conclude that protein methylation does not regulate the activity of NADPH-oxidases and that AMI-1 is a potent antioxidant with a greater potency than 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) and 4-hydroxy-2,2,6,6-tetramethylpiperydine-1-oxyl (Tempol).