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Osteoarthritis (OA) is a complex disease characterized by structural, functional, and metabolic deteriorations of the whole joint and periarticular tissues. In the current study, we aimed to investigate the possible effects of tempol on knee OA induced by the chemical chondrotoxic monosodium iodoacetate (MIA) which closely mimics both the pain and structural changes associated with human OA. Rats were administrated oral tempol (100 mg/kg) one week post-MIA injection (3 mg/50 μL saline) at the right knee joints for 21 consecutive days. Tempol improved motor performance and debilitated the MIA-related radiological and histological alterations. Moreover, it subsided the knee joint swelling. Tempol decreased the cartilage degradation-related biomarkers as matrix metalloproteinase-13, bone alkaline phosphatase (bone ALP), and fibulin-3. The superoxide dismutase mimetic effect of tempol was accompanied by decreased NADPH oxidase 4 (NOX4), inflammatory mediators, nuclear factor-kappa B (NF-κB), over-released transforming growth factor-β1 (TGF-β1). Tempol decreased the expression of chemokine (C-C motif) ligand 2 (CCL2). On the molecular level, tempol reduced the phosphorylated protein levels of p38 mitogen-activated protein kinase (MAPK), and small mother against decapentaplegic 3 homologs (SMAD3). These findings suggest the promising role of tempol in ameliorating MIA-induced knee OA in rats via collateral suppression of the catabolic signaling cascades including TGF-β1/SMAD3/NOX4, and NOX4/p38MAPK/NF-κB and therefore modulation of oxidative stress, catabolic inflammatory cascades, chondrocyte metabolic homeostasis.

HSV disease is distributed worldwide. Anti-herpesvirus drugs are a problem in clinical settings, particularly in immunocompromised individuals undergoing herpes simplex virus type 1 infection. In this work, 4-substituted-1,2,3-1H-1,2,3-triazole linked nitroxyl radical derived from TEMPOL were synthesized, and their ability to inhibit the in vitro replication of HSV-1 was evaluated. The nitroxide derivatives were characterized by infrared spectroscopy and elemental analysis, and three of them had their crystal structures determined by single-crystal X-ray diffraction. Four hybrid molecules showed important anti-HSV-1 activity with IC 50 values ranged from 0.80 to 1.32 µM. In particular, one of the nitroxide derivatives was more active than Acyclovir (IC 50  = 0.99 µM). All compounds tested were more selective inhibitors than the reference antiviral drug. Among them, two compounds were 4.5 (IC 50 0.80 µM; selectivity index CC 50 /IC 50 3886) and 7.7 times (IC 50 1.10 µM; selectivity index CC 50 /IC 50 6698) more selective than acyclovir (IC 50 0.99 µM; selectivity index CC 50 /IC 50 : 869). These nitroxide derivatives may be elected as leading compounds due to their antiherpetic activities and good selectivity., (© 2020. Springer Nature Switzerland AG.)

Nitric oxide (NO) metabolites have physiological and pharmacological importance and increasing their tissue concentrations may result in beneficial effects. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) has antioxidant properties that may improve NO bioavailability. Moreover, tempol increases oral nitrite-derived gastric formation of S-nitrosothiols (RSNO). We hypothesized that pretreatment with tempol may further increase tissue concentrations of NO-related species after oral nitrite administration and therefore we carried out a time-dependent analysis of how tempol affects the concentrations of NO metabolites in different tissues after oral nitrite administration to rats. NO metabolites (nitrate, nitrite and RSNO) were assessed by ozone-based reductive chemiluminescence assays in plasma, stomach, aorta, heart and liver samples obtained from anesthetized rats at baseline conditions and 15 min, 30 min, 2 h or 24 h after oral nitrite (15 mg/kg) was administered to rats pretreated with tempol (18 mg/kg) or vehicle 15 min prior to nitrite administration. Aortic protein nitrosation was assessed by resin-assited capture (SNO-RAC) method. We found that pretreatment with tempol transiently enhanced nitrite-induced increases in nitrite, RSNO and nitrate concentrations in the stomach and in the plasma (all P < 0.05), particularly for 15-30 min, without affecting aortic protein nitrosation. Pretreatment with tempol enhanced nitrite-induced increases in nitrite (but not RSNO or nitrate) concentrations in the heart (P < 0.05). In contrast, tempol attenuated nitrite-induced increases in nitrite, RSNO or nitrate concentrations in the liver. These findings show that pretreatment with tempol affects oral nitrite-induced changes in tissue concentrations of NO metabolites depending on tissue type and does not increase nitrite-induced vascular nitrosation. These results may indicate that oral nitrite therapy aiming at achieving increased nitrosation of cardiovascular targets requires appropriate doses of nitrite and is not optimized by tempol., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Decades of intense scientific research investigations clearly suggest that only a subset of a large number of metals, ceramics, polymers, composites, and nanomaterials are suitable as biomaterials for a growing number of biomedical devices and biomedical uses. However, biomaterials are prone to microbial infection due to Escherichia coli ( E. coli ), Staphylococcus aureus ( S. aureus ), Staphylococcus epidermidis ( S. epidermidis ), hepatitis, tuberculosis, human immunodeficiency virus (HIV), and many more. Hence, a range of surface engineering strategies are devised in order to achieve desired biocompatibility and antimicrobial performance in situ. Surface engineering strategies are a group of techniques that alter or modify the surface properties of the material in order to obtain a product with desired functionalities. There are two categories of surface engineering methods: conventional surface engineering methods (such as coating, bioactive coating, plasma spray coating, hydrothermal, lithography, shot peening, and electrophoretic deposition) and emerging surface engineering methods (laser treatment, robot laser treatment, electrospinning, electrospray, additive manufacturing, and radio frequency magnetron sputtering technique). Atomic-scale engineering, such as chemical vapor deposition, atomic layer etching, plasma immersion ion deposition, and atomic layer deposition, is a subsection of emerging technology that has demonstrated improved control and flexibility at finer length scales than compared to the conventional methods. With the advancements in technologies and the demand for even better control of biomaterial surfaces, research efforts in recent years are aimed at the atomic scale and molecular scale while incorporating functional agents in order to elicit optimal in situ performance. The functional agents include synthetic materials (monolithic ZnO, quaternary ammonium salts, silver nano-clusters, titanium dioxide, and graphene) and natural materials (chitosan, totarol, botanical extracts, and nisin). This review highlights the various strategies of surface engineering of biomaterial including their functional mechanism, applications, and shortcomings. Additionally, this review article emphasizes atomic scale engineering of biomaterials for fabricating antimicrobial biomaterials and explores their challenges.

The ability of sodium caprylate and l-menthol to fluidize phospholipid bilayers composed of lipids simulating the buccal epithelium was investigated using electron spin resonance (ESR) to evaluate the action of these agents as permeation enhancers. 5-Doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA) were used as spin labels to identify alterations in membrane fluidity near the polar head groups or inner acyl regions of the lipid bilayer, respectively. The molecular motion of both 5-DSA and 16-DSA showed increased disorder near the polar and inner hydrophobic regions of the bilayer in the presence of sodium caprylate suggesting fluidization in both the regions, which contributes to its permeation enhancing effects. L-menthol decreased the order parameter for 16-DSA, showing membrane fluidization only in the inner acyl regions of the bilayer, which also corresponded to its weaker permeation enhancing effects. The rapid evaluation of changes in fluidity of the bilayer in the presence of potential permeation enhancers using ESR enables improved selection of effective permeation enhancers and enhancer combinations based on their effect on membrane fluidization.

Introduction: The mechanical obstruction and pulmonary vasoconstriction are major determinants of the sudden right ventricular (RV) afterload increases observed during acute pulmonary thromboembolism (APT). Vasodilators and antioxidants agents have been shown to mitigate pulmonary hypertension. We examined whether sodium nitrite and the antioxidant tempol combination could be advantageous in an APT sheep model., Methods: APT was induced in anesthetized sheep by autologous blood clots (250 mg/kg) into the right atrium. Thirty minutes after APT induction, the animals received a continuous infusion of tempol (1.0 mg/kg/min), increasing sodium nitrite infusion (5, 15, and 50 μmol/kg), or a simultaneous combination of both drugs. Saline was used as a control treatment. Hemodynamic measurements were carried out every 15 min. Also, whole blood nitrite and serum 8-isoprostanes levels were measured., Results: APT induced sustained pulmonary hypertension, increased dp/dt max , and rate pressure product (RPP). Nitrite or tempol treatments attenuated these increases (P < 0.05). When both drugs were combined, we found a robust reduction in the RV RPP compared with the treatments alone (P < 0.05). The sole nitrite infusion increased blood nitrite concentrations by 35 ± 6 μM (P < 0.05), whereas the nitrite and tempol combination produced higher blood nitrite concentrations by approximately 54 ± 7 μM. Tempol or nitrite infusions, both alone or combined, blunted the increases in 8-isoprostane concentrations observed after APT., Conclusions: Nitrite and tempol combination protects against APT-induced RV wall stress. The association of both drugs may offer an advantage to treat RV failure during severe APT., (Copyright © 2021 Elsevier Inc. All rights reserved.)

COVID-19 is characterized by a dysregulation of inflammatory cytokines ultimately resulting a cytokine storm that can result in significant morbidity and mortality. We developed an in-vitro assay using activated peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS) or CD3 + CD28 to examine secretion of cytokines from antigen presenting cells (APCs) and T cells, respectively, in donor patients with a history of COVID-19 (convalescent) and uninfected negative controls. We hypothesized that a novel antioxidant called Tempol may decrease cytokines from activated peripheral blood cells from both COVID-19 patients and normal donors. Preincubation of immune cells with Tempol resulted in a significant (P < 0.05) decrease in multiple T cell and APC-derived cytokines from both cells of COVID-19 (n = 7) and uninfected donors (n = 7). These preliminary results suggest that Tempol has strong in-vitro anti-cytokine activity and supports additional studies examining the use of Tempol for the treatment of COVID-19., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Consumption of diet rich in fat and cigarette smoking (CS) are independent risk factors of non-alcoholic steatohepatitis (NASH), and they often occur together in some populations. The present study investigated the mechanisms of high-fat diet (HFD) and CS, individually and in combination, on the pathogenesis of NASH in mice. C57BL/6 male mice were subjected to either a low-fat chow (CH) or HFD with or without mainstream CS-exposure (4 cigarettes/day, 5 days/ week for 14 weeks). HFD alone caused hepatosteatosis (2.5-fold increase in TG content) and a significant increase in 3-nitrotyrisine (by ∼40-fold) but without an indication of liver injury, inflammation or fibrosis. CS alone in CH-fed mice increased in Tnfα expression and macrophage infiltration by 2-fold and relatively less increase in 3-nitrotyrosine (18-fold). Combination of HFD and CS precipitated hepatosteatosis to NASH reflected by exacerbated makers of liver inflammation and fibrosis which were associated with much severe liver oxidative stress (90-fold increase in 3-nitrotyrisine along with 6-fold increase in carbonylated proteins and 56% increase in lipid oxidations). Further studies were performed to administer the antioxidant tempol to CS exposed HFD mice and the results showed that the inhibition of liver oxidative stress prevented inflammatory and fibrotic changes in liver despite persisting hepatosteatosis. Our findings suggest that oxidative stress is a key mechanism underlying CS-promoted progression of simple hepatosteatosis to NASH. Targeting hepatic oxidative stress may be a viable strategy in halting the progression of metabolic associated fatty liver disease., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with low survival rate and a lack of biomarkers and targeted treatments. Here, we target pyruvate kinase M2 (PKM2), a key metabolic component of oncogenesis. In patients with TNBC, PKM2pS37 was identified as a prominent phosphoprotein corresponding to the aggressive breast cancer phenotype that showed a characteristic nuclear staining pattern and prognostic value. Phosphorylation of PKM2 at S37 was connected with a cyclin-dependent kinase (CDK) pathway in TNBC cells. In parallel, pyruvate kinase activator TEPP-46 bound PKM2pS37 and reduced its nuclear localization. In a TNBC mouse xenograft model, treatment with either TEPP-46 or the potent CDK inhibitor dinaciclib reduced tumor growth and diminished PKM2pS37. Combinations of dinaciclib with TEPP-46 reduced cell invasion, impaired redox balance, and triggered cancer cell death. Collectively, these data support an approach to identify PKM2pS37-positive TNBC and target the PKM2 regulatory axis as a potential treatment. SIGNIFICANCE: PKM2 phosphorylation marks aggressive breast cancer cell phenotypes and targeting PKM2pS37 could be an effective therapeutic approach for treating triple-negative breast cancer., (©2021 American Association for Cancer Research.)

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with a poor prognosis and limited treatment options. Oxidative and nitrosative stress is implicated as one of the main pathogenic pathways in IPF. The rationale for the use of antioxidants to treat lung fibrosis is appealing, however to date a consistent beneficial effect for such an approach has not been observed. We have recently demonstrated that nitroxides, particularly 3-carbamoyl-proxyl (3-CP), markedly reduce airway inflammation, airway hyper-responsiveness, and protein nitration of the lung tissue in a mouse model of ovalbumin-induced acute asthma, thus prompting its use for the treatment of IPF. The present study investigates the effect of 3-CP on the development of lung fibrosis using the murine intratracheal bleomycin model. 3-CP was administered either intranasally or orally during the entire experiment or starting 7 days after induction of the lung injury. 3-CP was found to be both a preventive and a therapeutic drug reducing the lung fibrosis (histological score), the increase in collagen content, protein nitration, TGF-β levels, the degree of weight loss as well as inhibiting the impairment of lung function. Nitroxides are catalytic antioxidants that preferentially detoxify radicals, and therefore the effect of 3-CP on the severity of the disease supports the involvement of reactive oxygen and nitrogen species in the disease pathology., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a potential redox agent in cells. The present study investigated changes in cellular reactive oxygen species (ROS) and glutathione (GSH) levels and in antioxidant enzymes, in Tempol-treated Calu-6 and A549 lung cancer cells, normal lung WI-38 VA-13 cells, and primary pulmonary fibroblasts. Results demonstrated that Tempol (0.5-4 mM) either increased or decreased general ROS levels in lung cancer and normal cells at 48 h and specifically increased O 2 •- levels in these cells. In addition, Tempol differentially altered the expression and activity of antioxidant enzymes such as superoxide dismutase, catalase, and thioredoxin reductase1 (TrxR1) in A549, Calu-6, and WI-38 VA-13 cells. In particular, Tempol treatment increased TrxR1 protein levels in these cells. Tempol at 1 mM inhibited the growth of lung cancer and normal cells by about 50% at 48 h but also significantly induced cell death, as evidenced by annexin V-positive cells. Furthermore, down-regulation of TrxR1 by siRNA had some effect on ROS levels as well as cell growth inhibition and death in Tempol-treated or -untreated lung cells. In addition, some doses of Tempol significantly increased the numbers of GSH-depleted cells in both cancer cells and normal cells at 48 h. In conclusion, Tempol differentially increased or decreased levels of ROS and various antioxidant enzymes in lung cancer and normal cells, and induced growth inhibition and death in all lung cells along with an increase in O 2 •- levels and GSH depletion., (© 2021. The Author(s).)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Objective: Adaptive immune resistance mediated by the cytokine interferon gamma (IFNG) still constitutes a major problem in cancer immunotherapy. We develop strategies for overcoming IFNG-mediated adaptive immune resistance in pancreatic ductal adenocarcinoma cancer (PDAC)., Design: We screened 429 kinase inhibitors for blocking IFNG-induced immune checkpoint (indoleamine 2,3-dioxygenase 1 (IDO1) and CD274) expression in a human PDAC cell line. We evaluated the ability of the cyclin-dependent kinase (CDK) inhibitor dinaciclib to block IFNG-induced IDO1 and CD274 expression in 24 human and mouse cancer cell lines as well as in primary cancer cells from patients with PDAC or ovarian carcinoma. We tested the effects of dinaciclib on IFNG-induced signal transducer and activator of transcription 1 activation and immunological cell death, and investigated the potential utility of dinaciclib in combination with IFNG for pancreatic cancer therapy in vivo, and compared gene expression levels between human cancer tissues with patient survival times using the Cancer Genome Atlas datasets., Results: Pharmacological (using dinaciclib) or genetic (using shRNA or siRNA) inactivation of CDK1/2/5 not only blocks JUN-dependent immune checkpoint expression, but also triggers histone-dependent immunogenic cell death in immortalised or primary cancer cells in response to IFNG. This dual mechanism turns an immunologically 'cold' tumour microenvironment into a 'hot' one, dramatically improving overall survival rates in mouse pancreatic tumour models (subcutaneous, orthotopic and transgenic models). The abnormal expression of CDK1/2/5 and IDO1 was associated with poor patient survival in several cancer types, including PDAC., Conclusion: CDK1/2/5 kinase activity is essential for IFNG-mediated cancer immunoevasion. CDK1/2/5 inhibition by dinaciclib provides a novel strategy to overcome IFNG-triggered acquired resistance in pancreatic tumour immunity., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Medulloblastoma (MB) is the most common aggressive paediatric brain tumour and, despite the recent progress in the treatments of MB patients, there is still an urgent need of complementary or alternative therapeutic options for MB infants. Cyclin Dependent Kinase inhibitors (CDKi) are at the front-line of novel targeted treatments for multiple cancers and the CDK4/6 specific inhibitor palbociclib has been pre-clinically identified as an effective option for MB cells. Herein, we identified the pan-CDKi dinaciclib as a promising alternative to palbociclib for the suppression of MB cells proliferation. We present evidence supporting dinaciclib's ability to inhibit MB cells in vitro proliferation at considerably lower doses than palbociclib. Sequencing data and pathway analysis suggested that dinaciclib is a potent cell death inducer in MB cells. We found that dinaciclib-triggered apoptosis is triggered by CDK9 inhibition and the resultant reduction in RNA pol II phosphorylation, which leads to the downregulation of the oncogenic marker MYC, and the anti-apoptotic protein MCL-1. Specifically, we demonstrated that MCL-1 is a key apoptotic mediator for MB cells and co-treatment of dinaciclib with BH3 mimetics boosts the therapeutic efficacy of dinaciclib. Together, these findings highlight the potential of multi-CDK inhibition by dinaciclib as an alternative option to CDK4/6 specific inhibition, frequently associated with drug resistance in patients.

Background: In the present study, we examined superoxide-mediated excitatory nociceptive transmission on at-level neuropathic pain following spinal thoracic 10 contusion injury (SCI) in male Sprague Dawley rats., Methods: Mechanical sensitivity at body trunk, neuronal firing activity, and expression of superoxide marker/ionotropic glutamate receptors (iGluRs)/CamKII were measured in the T7/8 dorsal horn, respectively., Results: Topical treatment of superoxide donor t-BOOH (0.4 mg/kg) increased neuronal firing rates and pCamKII expression in the naïve group, whereas superoxide scavenger Tempol (1 mg/kg) and non-specific ROS scavenger PBN (3 mg/kg) decreased firing rates in the SCI group (* p < 0.05). SCI showed increases of iGluRs-mediated neuronal firing rates and pCamKII expression (* p < 0.05); however, t-BOOH treatment did not show significant changes in the naïve group. The mechanical sensitivity at the body trunk in the SCI group (6.2 ± 0.5) was attenuated by CamKII inhibitor KN-93 (50 μg, 3.9 ± 0.4) or Tempol (1 mg, 4 ± 0.4) treatment (* p < 0.05). In addition, the level of superoxide marker Dhet showed significant increase in SCI rats compared to the sham group (11.7 ± 1.7 vs. 6.6 ± 1.5, * p < 0.05)., Conclusions: Superoxide and the pCamKII pathway contribute to chronic at-level neuropathic pain without involvement of iGluRs following SCI.

Background: Sperm mitochondria are the major site of reactive oxygen species (ROS) production and excess production during freezing-thawing process inflicts oxidative damages to spermatozoa. Buffalo spermatozoa are more prone to oxidative damage due to inherently more polyunsaturated fatty acids and low cholesterol to phospholipids ratio in the plasma membrane. A mitochondrial targeted antioxidant, Mito-TEMPO was used in this study., Objective: To study the effect of Mito-TEMPO incorporated semen extender on the post-thaw semen quality in buffalo., Materials and Methods: A total of 18 ejaculates from three murrah buffalo bulls with ≥70% individual progressive motility were utilized for the study. Each semen sample was equally divided and extended with five groups: Group I (Control, without Mito-TEMPO addition); Group II (10 µM Mito-TEMPO); Group III (50 µM Mito-TEMPO); Group IV (100 µM Mito-TEMPO); Group V (500 µM Mito-TEMPO) to have 80×10 6 progressive motile sperm/mL of extender, filled and sealed in French mini straws (0.25 mL) and frozen following equilibration. The effect of Mito-TEMPO was assessed at fresh/post-dilution and post-thaw stages by evaluating physico-morphological attributes and functional membrane integrity such as hypo-osmotic swelling test (HOST)., Results: Initial progressive motility, viability, acrosomal integrity and HOS response was significantly (p<0.05) improved and sperm abnormality was significantly (p<0.05) reduced in extended semen with Mito-TEMPO (50 µM) compared to control at post-thaw stage, although improvement was also observed at 10 and 100 µM in post-thaw samples., Conclusion: Mito-TEMPO incorporated semen extender at 50 µM concentration, could be part of a rationale for improving post-thaw semen quality in buffalo.

Background: Alhtough anti-HER2 tyrosine kinase inhibitors (TKIs) have radically prolonged survival and improved prognosis in HER2-positive breast cancer patients, resistance to these therapies is a constant obstacle leading to TKIs treatment failure and tumour progression., Methods: To develop new strategies to enhance TKIs efficiency by combining synergistic gene targets, we performed panel library screening using the CRISPR/Cas9 knockout technique based on data mining across TCGA data sets and verified the candidate target in preclinical models and breast cancer high-throughput sequencing data sets., Results: We identified that CDK12, co-amplified with HER2 in a high frequency, is powerful to sensitise or resensitise HER2-positive breast cancer to anti-HER2 TKIs lapatinib, evidenced by patient-derived organoids in vitro and cell-derived xenograft or patient-derived xenograft in vivo. Exploring mechanisms, we found that inhibition of CDK12 attenuated PI3K/AKT signal, which usually serves as an oncogenic driver and is reactivated when HER2-positive breast cancers develop resistance to lapatinib. Combining CDK12 inhibition exerted additional suppression on p-AKT activation induced by anti-HER2 TKIs lapatinib treatment. Clinically, via DNA sequencing data for tumour tissue and peripheral blood ctDNA, we found that HER2-positive breast cancer patients with CDK12 amplification responded more insensitively to anti-HER2 treatment than those without accompanying CDK12 amplification by harbouring a markedly shortened progression-free survival (PFS) (median PFS: 4.3 months versus 6.9 months; hazards ratio [HR] = 2.26 [95% confidence interval [CI] = 1.32-3.86]; P = 0.0028)., Conclusions: Dual inhibition of HER2/CDK12 will prominently benefit the outcomes of patients with HER2-positive breast cancer by sensitising or resensitising the tumours to anti-HER2 TKIs treatment., Competing Interests: Conflict of interest statement The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Herein, we report the neuroprotective and antioxidant activity of 1,1'-biphenyl nitrones ( BPNs ) 1 - 5 as α-phenyl- N - tert -butylnitrone analogues prepared from commercially available [1,1'-biphenyl]-4-carbaldehyde and [1,1'-biphenyl]-4,4'-dicarbaldehyde. The neuroprotection of BPNs 1 - 5 has been measured against oligomycin A/rotenone and in an oxygen-glucose deprivation in vitro ischemia model in human neuroblastoma SH-SY5Y cells. Our results indicate that BPNs 1 - 5 have better neuroprotective and antioxidant properties than α-phenyl- N - tert -butylnitrone ( PBN ), and they are quite similar to N -acetyl-L-cysteine ( NAC ), which is a well-known antioxidant agent. Among the nitrones studied, homo-bis-nitrone BPHBN5 , bearing two N - tert -Bu radicals at the nitrone motif, has the best neuroprotective capacity (EC 50 = 13.16 ± 1.65 and 25.5 ± 3.93 μM, against the reduction in metabolic activity induced by respiratory chain blockers and oxygen-glucose deprivation in an in vitro ischemia model, respectively) as well as anti-necrotic, anti-apoptotic, and antioxidant activities (EC 50 = 11.2 ± 3.94 μM), which were measured by its capacity to reduce superoxide production in human neuroblastoma SH-SY5Y cell cultures, followed by mononitrone BPMN3 , with one N -Bn radical, and BPMN2 , with only one N - tert -Bu substituent. The antioxidant activity of BPNs 1 - 5 has also been analyzed for their capacity to scavenge hydroxyl free radicals (82% at 100 μM), lipoxygenase inhibition, and the inhibition of lipid peroxidation (68% at 100 μM). Results showed that although the number of nitrone groups improves the neuroprotection profile of these BPNs , the final effect is also dependent on the substitutent that is being incorporated. Thus, BPNs bearing N - tert -Bu and N -Bn groups show better neuroprotective and antioxidant properties than those substituted with Me. All these results led us to propose homo- bis -nitrone BPHBN5 as the most balanced and interesting nitrone based on its neuroprotective capacity in different neuronal models of oxidative stress and in vitro ischemia as well as its antioxidant activity.

Human epidermal growth factor receptor 2 gene (HER2) is focally amplified in approximately 20% of breast cancers. HER2 inhibitors alone are not effective, and sensitizing agents will be necessary to move away from a reliance on heavily toxic chemotherapeutics. We recently demonstrated that the efficacy of HER2 inhibitors is mitigated by uniformly low levels of the myeloid cell leukemia 1 (MCL-1) endogenous inhibitor, NOXA. Emerging clinical data have demonstrated that clinically advanced cyclin-dependent kinase (CDK) inhibitors are effective MCL-1 inhibitors in patients, and, importantly, well tolerated. We, therefore, tested whether the CDK inhibitor, dinaciclib, could block MCL-1 in preclinical HER2-amplified breast cancer models and therefore sensitize these cancers to dual HER2/EGFR inhibitors neratinib and lapatinib, as well as to the novel selective HER2 inhibitor tucatinib. Indeed, we found dinaciclib suppresses MCL-1 RNA and is highly effective at sensitizing HER2 inhibitors both in vitro and in vivo. This combination was tolerable in vivo. Mechanistically, liberating the effector BCL-2 protein, BAK, from MCL-1 results in robust apoptosis. Thus, clinically advanced CDK inhibitors may effectively combine with HER2 inhibitors and present a chemotherapy-free therapeutic strategy in HER2-amplified breast cancer, which can be tested immediately in the clinic.

Vitamin D modulates about 3% human gene transcription besides the classical action on calcium/phosphorus homeostasis. The blood pressure-lowing and other protective action on cardiovascular disease have been reported. The present study aims to examine whether COX-1 and COX-2 were implicated in endothelial dysfunction in hypertension and calcitriol, an active form of vitamin D preserved endothelial function through regulating COX expression. Isometric study demonstrated the impaired endothelium-dependent relaxation (EDR) in renal arteries from spontaneously hypertensive rats were reversed by 12 h-calcitriol treatment and COX-1 and COX-2 inhibitors. Combined uses of COX-1 and COX-2 inhibitor induced more improved relaxations. Exaggerated expressions of COX-1 and COX-2 in renal artery from SHR were inhibited by 12 h-administration of calcitriol, NADPH oxidase inhibitor DPI, or reactive oxygen species (ROS) scavenger tempol. Furthermore, in normotensive WKY rats, calcitriol prevents against the blunted EDR in renal arteries by 12 h-Ang II exposure, with similar improvements by COX-1 and COX-2 inhibitors. Accordingly, increased COX-1 and COX-2 expressions by Ang II exposure were corrected by losartan, DPI, or tempol. Studies on human renal artery also revealed the beneficial action of calcitriol is mediated by suppressing COX-1 and COX-2 expressions, dependent on vitamin D receptor (VDR) activation. Taken together, our findings showed that COX-1 and COX-2 are positively involved in the renovascular dysfunction in hypertension and via VDR, calcitriol benefits renovasular function by suppressing COX-1 and COX-2 expressions. Furthermore, ROS is involved in the COX-1 and COX-2 up-regulations of renal arteries, maybe serving as a mediator in the inhibitory action of calcitriol on COX expression.

Oxidative stress is a critical element in relationship to the pathophysiology of Duchenne muscular dystrophy (DMD). In the mice the diaphragm (DIA) is most resembles the dystrophic human pathology. In this study we have evaluated the consequences of a synthetic antioxidant (tempol) on oxidative stress parameters in the DIA muscle of mdx mice. The mdx mice were separated into two groups: mdx, the control group receiving intraperitoneal (i.p.) injections of saline solution (100 µL), and mdxT, the treated group receiving i.p. injections of tempol (100 mg/kg). The tempol-treated group showed reduced oxidative stress markers, decreasing the dihydroethidium reaction (DHE) area; autofluorescent lipofuscin granules; and 4-hydroxynonenal (4-HNE)-protein adduct levels. DIA muscle of mdx mice. At the same time, the manganese-superoxide dismutase 2 (SOD2) levels were increased in the tempol-treated group. In addition, the tempol-treated group showed reduced levels of glutathione-disulphide reductase (GSR), glutathione peroxidase 1 (GPx1) and catalase (CAT) in immunoblots. The tempol-treated group has also shown lower relative gene expression of SOD1, CAT and GPx than the non-treated group. Our data demonstrated that tempol treatment reduced oxidant parameters and increased anti-oxidant SOD2 levels in the DIA muscle of mdx mice, which may contribute to the normalization of the redox homeostasis of dystrophic muscles., (© 2020 Company of the International Journal of Experimental Pathology (CIJEP).)

Methamphetamine (METH) is a powerful psychostimulant that is causing serious health problems worldwide owing to imprudent abuses. Recent studies have suggested that METH has deleterious effects on the blood-brain barrier (BBB). A few studies have also been conducted on the mechanisms whereby METH-induced oxidative stress causes BBB dysfunction. We investigated whether N-tert-butyl-α-phenylnitrone (PBN) has protective effects on BBB function against METH exposure in primary human brain microvascular endothelial cells (HBMECs). We found that METH significantly increased reactive oxygen species (ROS) generation in HBMECs. Pretreatment with PBN decreased METH-induced ROS production. With regard to BBB functional integrity, METH exposure elevated the paracellular permeability and reduced the monolayer integrity; PBN treatment reversed these effects. An analysis of the BBB structural properties, by immunostaining junction proteins and cytoskeleton in HBMECs, indicated that METH treatment changed the cellular localization of the tight (ZO-1) and adherens junctions (VE-cadherin) from the membrane to cytoplasm. Furthermore, METH induced cytoskeletal reorganization via the formation of robust stress fibers. METH-induced junctional protein redistribution and cytoskeletal reorganization were attenuated by PBN treatment. Our results suggest that PBN can act as a therapeutic reagent for METH-induced BBB dysfunction by inhibiting excess ROS generation.

A rise in heart disease incidence in women after menopause has led to investigations into the role of female sex hormones on cardiac function. Although various adverse changes in cardiac contractile function following loss of female sex hormones have been reported, a clear mechanism of action has never been characterized. In order to examine whether an elevation in oxidative stress is a major cause of cardiac contractile dysfunction after female sex hormone deprivation, cardiac functions of ovariectomized rats with and without supplementation of superoxide scavenger tempol were compared to those of sham-operated controls. Chronic deprivation of female sex hormones reduced total oxidative capacity and increased plasma carbonyl protein content. Tempol supplementation of ovariectomized rats significantly ameliorated plasma oxidative stress status. Echocardiography demonstrated a significant decrease in left ventricular ejection fraction in ovariectomized rats, which was completely prevented by tempol supplementation. Decreased myocardial contractility occurs with reduced maximum myofilament force of contraction and amplitude of transient intracellular Ca 2+ concentration, both phenomena completely attenuated by tempol supplementation. However, tempol only partially prevented shift of heart myosin heavy chain from dominant α-to β-isoform of ovariectomized rats. Immunoblot analysis of protein carbonylation indicated that tempol supplementation significantly reduced the level of cardiac myofibrillar proteins oxidation increased in ovariectomized rat heart. Taken together, the results indicate changes of cardiac contractile machinery following loss of female sex hormones were, in part, due to an increase in oxidative stress, and antioxidant supplementation could be considered another potential prevention measure in postmenopausal women., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Derivatives of tirapazamine and other heteroaromatic N- oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities, which are typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the role of NAD(P)H:quinone oxidoreductase (NQO1) in ArN→O aerobic cytotoxicity. We synthesized 9 representatives of ArN→O with uncharacterized redox properties and examined their single-electron reduction by rat NADPH:cytochrome P-450 reductase (P-450R) and Plasmodium falciparum ferredoxin:NADP + oxidoreductase ( Pf FNR), and by rat NQO1. NQO1 catalyzed both redox cycling and the formation of stable reduction products of ArN→O. The reactivity of ArN→O in NQO1-catalyzed reactions did not correlate with the geometric average of their activity towards P-450R- and Pf FNR, which was taken for the parameter of their redox cycling efficacy. The cytotoxicity of compounds in murine hepatoma MH22a cells was decreased by antioxidants and the inhibitor of NQO1, dicoumarol. The multiparameter regression analysis of the data of this and a previous study (DOI: 10.3390/ijms20184602) shows that the cytotoxicity of ArN→O ( n = 18) in MH22a and human colon carcinoma HCT-116 cells increases with the geometric average of their reactivity towards P-450R and Pf FNR, and with their reactivity towards NQO1. These data demonstrate that NQO1 is a potentially important target of action of heteroaromatic N- oxides.

Background: The interaction between proteins and nanoparticles is a very relevant subject because of the potential applications in medicine and material science in general. Further interest derives from the amyloidogenic character of the considered protein, β2-microglobulin (β2m), which may be regarded as a paradigmatic system for possible therapeutic strategies. Previous evidence showed in fact that gold nanoparticles (AuNPs) are able to inhibit β2m fibril formation in vitro., Methods: NMR (Nuclear Magnetic Resonance) and ESR (Electron Spin Resonance) spectroscopy are employed to characterize the paramagnetic perturbation of the extrinsic nitroxide probe Tempol on β2m in the absence and presence of AuNPs to determine the surface accessibility properties and the occurrence of chemical or conformational exchange, based on measurements conducted under magnetization equilibrium and non-equilibrium conditions., Results: The nitroxide perturbation analysis successfully identifies the protein regions where protein-protein or protein-AuNPs interactions hinder accessibility or/and establish exchange contacts. These information give interesting clues to recognize the fibrillation interface of β2m and hypothesize a mechanism for AuNPs fibrillogenesis inhibition., Conclusions: The presented approach can be advantageously applied to the characterization of the interface in protein-protein and protein-nanoparticles interactions.

Background: Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium., Objective: We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy., Study Design: Female p70S6K1 heterogeneous knockout (p70S6K1 +/- ) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1 +/- ) mice to generate wild-type (WT), p70S6K1 +/- and p70S6K1 knockout (p70S6K1 -/- ) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For in vitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions., Results: Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes in vivo and high glucose in vitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation., Conclusion: The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cerebral cavernous malformations (CCMs) are ectatic capillary-venous malformations that develop in approximately 0.5% of the population. Patients with CCMs may develop headaches, focal neurologic deficits, seizures, and hemorrhages. While symptomatic CCMs, depending upon the anatomic location, can be surgically removed, there is currently no pharmaceutical therapy to treat CCMs. Several mouse models have been developed to better understand CCM pathogenesis and test therapeutics. The most common mouse models induce a large CCM burden that is anatomically restricted to the cerebellum and contributes to lethality in the early days of life. These inducible models thus have a relatively short period for drug administration. We developed an inducible CCM3 mouse model that develops CCMs after weaning and provides a longer period for potential therapeutic intervention. Using this new model, three recently proposed CCM therapies, fasudil, tempol, vitamin D 3 , and a combination of the three drugs, failed to substantially reduce CCM formation when treatment was administered for 5 weeks, from postnatal day 21 (P21) to P56. We next restricted Ccm3 deletion to the brain vasculature and provided greater time (121 days) for CCMs to develop chronic hemorrhage, recapitulating the human lesions. We also developed the first model of acute CCM hemorrhage by injecting mice harboring CCMs with lipopolysaccharide. These efficient models will enable future drug studies to more precisely target clinically relevant features of CCM disease: CCM formation, chronic hemorrhage, and acute hemorrhage.

The heart is sensitive to oxidative damage but a global view on how the cardiac proteome responds to oxidative stressors has yet to fully emerge. Using quantitative tandem mass spectrometry, we assessed the effects of acute exposure of the oxidative stress inducer paraquat on protein expression in mouse hearts. We observed widespread protein expression changes in the paraquat-exposed heart especially in organelle-containing subcellular fractions. During cardiac response to acute oxidative stress, proteome changes are consistent with a rapid reduction of mitochondrial metabolism, coupled with activation of multiple antioxidant proteins, reduction of protein synthesis and remediation of proteostasis. In addition to differential expression, we saw evidence of spatial reorganizations of the cardiac proteome including the translocation of hexokinase 2 to more soluble fractions. Treatment with the antioxidants Tempol and MitoTEMPO reversed many proteomic signatures of paraquat but this reversal was incomplete. We also identified a number of proteins with unknown function in the heart to be triggered by paraquat, suggesting they may have functions in oxidative stress response. Surprisingly, protein expression changes in the heart correlate poorly with those in the lung, consistent with differential sensitivity or stress response in these two organs. The results and data set here could provide insights into oxidative stress responses in the heart and avail the search for new therapeutic targets.

Cholangiocarcinoma (CCA) is a highly invasive cancer, diagnosed at an advanced stage, and refractory to surgical intervention and chemotherapy. Cyclin-dependent kinases (CDKs) regulate cell cycle progression and transcriptional processes, and are considered potential therapeutic targets for cancer. Dinaciclib is a small molecule multi-CDK inhibitor targeting CDK 2/5/9. In this study, the therapeutic efficacy of dinaciclib was assessed using patient-derived xenograft cells (PDXC) and CCA cell lines. Treatment with dinaciclib significantly suppressed cell proliferation, induced caspase 3/7 levels and apoptotic activity in PDXC and CCA cell lines. Dinaciclib suppressed expression of its molecular targets CDK2/5/9, and anti-apoptotic BCL-XL and BCL2 proteins. Despite the presence of cyclin D1 amplification in the PDXC line, palbociclib treatment had no effect on cell proliferation, cell cycle or apoptosis in the PDXC as well as other CCA cell lines. Importantly, dinaciclib, in combination with gemcitabine, produced a robust and sustained inhibition of tumor progression in vivo in a PDX mouse model, greater than either of the treatments alone. Expression levels of two proliferative markers, phospho-histone H3 and Ki-67, were substantially suppressed in samples treated with the combination regimen. Our results identify dinaciclib as a novel and potent therapeutic agent alone or in combination with gemcitabine for the treatment of CCA.

The hormonal luminal-A is the most pre-dominant sub type of breast cancer (BC), and it is associated with a high level of cyclin D1 in Saudi patients. Tamoxifen is the golden therapy for hormonal BC, but resistance of cancer cells to tamoxifen contributes to the recurrence of BC due to many reasons, including high levels of AIB1 and cyclin D1. Overcoming drug resistance could be achieved by exploring alternative targetable therapeutic pathways and new drugs or combinations. The objective of this study was to determine the differentially enriched pathways in 12 samples of Saudi women diagnosed with luminal-A using the PamChip peptide microarray-based kinase activity profiling, and to compare the activity of HAA 2020 and dinaciclib with tamoxifen in singles and combinations in the MCF7 luminal-A cell line. Our results of network and pathway analysis of the 12 samples highlighted the importance of VEGFR and CDKs in promoting luminal-A breast cancer. The activation of VEGF signaling via VEGFR-2 leads to activation of PI3K/AKT kinases and an increase of cell survival, and leads to activation of Hsp90, which induces the phosphorylation of FAK1, resulting in cytoskeleton remodeling. PLC-gamma 1 is also activated, leading to FAK-2 and PKC activation. Notably, the G 1 /S cell cycle phases and phosphorylation processes contribute to the top seven tumorigenesis processes in the 12 samples. Further, the MTT combination of HAA 2020 and dinaciclib showed the best combination index (CI), was more clonogenic against MCF7 cells compared to the other combinations, and it also showed the best selectivity index (SI) in normal MRC5 cells. Interestingly, HAA 2020 and dinaciclib showed a synergistic apoptotic and G 1 cell cycle effect in MCF7 cells, which was supported by their synergistic CDK2, cyclin D1, and PCNA inhibition activities. Additionally, the combination showed VEGFR-2 and Hsp90 inhibition activities in MCF7 cells. The results show the significance of targeting VEGFR-2 and cyclin D1 in Saudi luminal-A breast cancer patients, and the effect of combining HAA 2020 and dinaciclib on those targets in the MCF7 model. It also warrants further preclinical and in vivo investigations for the combination of HAA 2020 and dinaciclib as a possible future second-line treatment for luminal-A breast cancers., Competing Interests: Supplementary Materials: The following are available online at www.mdpi.com/1420-3049/25/20/4606/s1, Figure S1: Key of pathway analysis symbols, Table S2: Enrichment analysis by GO processes, Excel file S3: Breast phosphorylation by tyrosine kinase (PTK) and serine/threonine kinase (STK) data, Figure S4: Histograms of cell cycle analysis in MCF7 cells, Figure S5: Histograms showing detection of apoptosis in MCF7 cells (24 h).

Background and aims : Lipopolysaccharide (LPS), an endotoxin, is a component of the outer membrane of Gram-negative bacteria that is able to activate the peripheral immune system, leading to changes in signalling pathways that act locally and systemically to achieve adaptive responses. Sickness behaviour is a motivational state in response to endotoxin exposure and includes depressed activity and a reduction of exploratory behaviour, potentially reorganising organism priorities to cope with infectious diseases. We hypothesised that 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) modulates the leukocyte response to endotoxins and decreases LPS-induced sickness behaviour in mice. Methods : The effects of Tempol on LPS-induced peritonitis and the respiratory burst of neutrophils primed with LPS and triggered by phorbol 12-myristate-13-acetate (PMA) were evaluated. To evaluate the effects of Tempol on sickness behaviour, the mice were submitted to an open field and forced swim tests. Results : Tempol (50-100   μM/10 6  cells) decreased the respiratory burst of LPS-primed and PMA-stimulated neutrophils in vitro . In vivo , this nitroxide (30 and 100   mg/kg body weight) inhibited leukocyte migration to the peritoneal cavity after LPS administration in mice. Moreover, Tempol pretreatment (30 and 100   mg/kg body weight) before LPS administration also attenuated sickness behavioural changes. Conclusions : Together, these findings shed light on the mechanisms underlying the anti-inflammatory potential and confirm the therapeutic potential of nitroxides.

Renal proximal tubular apoptosis plays a critical role in kidney health and disease. However, cellular molecules that trigger renal apoptosis remain elusive. Here, we evaluated the effect of inhibiting protein disulfide isomerase (PDI), a critical thioredoxin chaperone protein, on apoptosis as well as the underlying mechanisms in human renal proximal tubular (HK2) cells. HK2 cells were transfected with PDI-specific siRNA in the absence and presence of an antioxidant, tempol. PDI siRNA transfection resulted in a decrease of ~70% in PDI protein expression and enzyme activity. PDI inhibition increased caspase-3 activity and induced profound cell apoptosis. Mitochondrial function, as assessed by mitochondrial cytochrome c levels, mitochondrial membrane potential, oxygen consumption, and ATP levels, was significantly reduced in PDI-inhibited cells. Also, PDI inhibition caused nuclear factor erythroid 2-related factor 2 (Nrf2; a redox-sensitive transcription factor) cytoplasmic sequestration, decreased superoxide dismutase and glutathione- S -transferase activities, and increased oxidative stress. In PDI-inhibited cells, tempol reduced apoptosis, caspase-3 activity, and oxidative stress and also restored Nrf2 nuclear translocation and mitochondrial function. Silencing Nrf2 in the cells abrogated the beneficial effect of tempol, whereas Kelch-like ECH-associated protein 1 (an Nrf2 regulatory protein) silencing protected cells from PDI inhibitory effects. Collectively, our data indicate that PDI inhibition diminishes Nrf2 nuclear translocation, causing oxidative stress that further triggers mitochondrial dysfunction and renal cell apoptosis. This study suggests an important role for PDI in renal cell apoptosis involving Nrf2 and mitochondrial dysfunction.

Cigarette smoke is a complex mixture capable of triggering inflammation and oxidative damage in animals at pulmonary and systemic levels. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) reduces tissue injury associated with inflammation in vivo by mechanisms that are not completely understood. Here we evaluated the effect of tempol on inflammation and oxidative damage induced by acute exposure to cigarette smoke in vivo. Male C57BL/6 mice (n = 32) were divided into 4 groups (n = 8 each): 1) control group exposed to ambient air (GC), 2) animals exposed to cigarette smoke for 5 days (CSG), mice treated 3) prior or 4) concomitantly with tempol (50 mg/kg/day) and exposed to cigarette smoke for 5 days. The results showed that the total number of leukocytes and neutrophils increased in the respiratory tract and lung parenchyma of mice exposed to cigarette smoke. Likewise, MPO levels and activity as well as lipid peroxidation and lung protein nitration and carbonylation also increased. Administration of tempol before or during exposure to cigarette smoke inhibited all the above parameters. Tempol also reduced the pulmonary expression of the inflammatory cytokines Il-6, Il-1β and Il-17 to basal levels and of Tnf-α by approximately 50%. In contrast, tempol restored Il-10 and Tgf-β levels and enhanced the expression of Nrf2-associated genes, such as Ho-1 and Gpx2. Accordingly, total GPx activity increased in lung homogenates of tempol-treated animals. Taken together, our results show that tempol protects mouse lungs from inflammation and oxidative damage resulting from exposure to cigarette smoke, likely through reduction of leukocyte infiltration and increased transcription of some of the Nrf2-controlled genes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study investigated whether nitroxide radical (4-amino-TEMPOL)-containing nanoparticles (RNPs; antioxidant nanomedicine) can prevent neurovascular unit impairment caused by reactive oxygen species (ROS) after cerebral ischemia-reperfusion. C57BL/6J mice underwent transient middle cerebral artery occlusion (tMCAO). The mice were randomly divided and administered intra-arterial RNPs injection (9 mg/kg, 7 μM/kg), edaravone (3 mg/kg, 17 μM/kg), or phosphate-buffered saline (control group). Survival rate and neurological score were evaluated 24 h post-injection. RNPs distribution was determined using immunofluorescence staining and blood-brain barrier (BBB) disruption using Evans blue extravasation assay. Effect of RNPs and edaravone on microglia polarization into microglia M1 and M2 was evaluated. We also determined multiple ROS-scavenging activities in brain homogenates of RNPs- and edaravone-treated animals using an electron spin resonance-based spin-trapping method. Compared with edaravone, RNPs significantly improved the survival rate and neurological deficit, inhibited BBB disruption and supported polarization of microglia into M2 microglia. RNPs were localized in endothelial cells, the perivascular space, neuronal cell cytoplasm, astrocytes, and microglia. Scavenging capacities of hydroxyl, alkoxyl, and peroxyl radicals were significantly higher in the RNPs-treated group. RNPs show promising results as a future neuroprotective nanomedicine approach for cerebral ischemia-reperfusion injury., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Regulated necrosis has been reported to exert an important role in the pathogenesis of various diseases, including renal ischemia-reperfusion (I/R) injury. Damage to renal tubular epithelial cells and subsequent cell death initiate the progression of acute kidney injury (AKI) and subsequent chronic kidney disease (CKD). We found that ferroptosis appeared in tubular epithelial cells (TECs) of various human kidney diseases and the upregulation of tubular proferroptotic gene ACSL4 was correlated with renal function in patients with acute kidney tubular injury. XJB-5-131, which showed high affinity for TECs, attenuated I/R-induced renal injury and inflammation in mice by specifically inhibiting ferroptosis rather than necroptosis and pyroptosis. Single-cell RNA sequencing (scRNA-seq) indicated that ferroptosis-related genes were mainly expressed in tubular epithelial cells after I/R injury, while few necroptosis- and pyroptosis-associated genes were identified to express in this cluster of cell. Taken together, ferroptosis plays an important role in renal tubular injury and the inhibition of ferroptosis by XJB-5-131 is a promising therapeutic strategy for protection against renal tubular cell injury in kidney diseases.

CVT-313 is a potent CDK2 inhibitor that was identified by screening a purine-analogue library and is currently in preclinical studies. Since this molecule has the potential to be developed as a CDK2 inhibitor for cancer therapy, the potency of CVT-313 to bind and stabilize CDK2 was evaluated, together with its ability to inhibit aberrant cell proliferation. CVT-313 increased the melting temperature of CDK2 by 7°C in thermal stabilization studies, thus indicating its protein-stabilizing effect. CVT-313 inhibited the growth of human lung carcinoma cell line A549 in a dose-dependent manner, with an IC 50 of 1.2 µM, which is in line with the reported biochemical potency of 0.5 µM. To support the further chemical modification of CVT-313 and to improve its biochemical and cellular potency, a crystal structure was elucidated in order to understand the molecular interaction of CVT-313 and CDK2. The crystal structure of CDK2 bound to CVT-313 was determined to a resolution of 1.74 Å and clearly demonstrated that CVT-313 binds in the ATP-binding pocket, interacting with Leu83, Asp86 and Asp145 directly, and the binding was further stabilized by a water-mediated interaction with Asn132. Based on the crystal structure, further modifications of CVT-313 are proposed to provide additional interactions with CDK2 in the active site, which may significantly increase the biochemical and cellular potency of CVT-313.

Aims: Vascular dysfunction plays a key role in sepsis but the role of perivascular adipose tissue (PVAT) in this condition is relatively unknown., Main Methods: Sepsis was induced by cecal ligation and puncture (CLP). The responses of the aorta and superior mesenteric artery to norepinephrine in the presence or absence of PVAT were evaluated. Fluorescent probes measured the production of nitric oxide (NO) and reactive oxygen species (ROS). NO synthases (NOS) and β3-adrenoceptor expression were detected by immunofluorescence and S-nitrosylation by the biotin switch assay., Key Findings: Aorta and superior mesenteric arteries from septic animals with intact PVAT showed a worsened response to the vasoconstrictor compared to vessels without PVAT. PVAT from the aorta (APVAT) produced NO and ROS whereas PVAT from the superior mesenteric artery (MPVAT) produced only ROS. Septic APVAT exhibited a higher density of NOS-1 and NOS-3. S-nitrosylation was found in APVAT. Donor (PVAT obtained from normal or septic rats):Host (normal vessel without PVAT) experiments showed that L-NAME, ODQ and β3-adrenergic receptor antagonist blocked the septic APVAT anti-contractile effect. None of these compounds affected MPVAT; tempol, but not apocynin, blocked its anti-contractile effect., Significance: PVAT contributes to the anti-contractile effect in the aorta and mesenteric artery of septic rats through different pathways. β3-Adrenergic receptor and NO appear to be key mediators of this effect in APVAT, but not in MPVAT where ROS seem to be a relevant mediator. Therefore, PVAT is a relevant player of sepsis vascular dysfunction., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: To investigate whether toll-like receptor 7 (TLR7) activation induces an increase in blood pressure and vascular damage in wild-type mice treated with the TLR7 agonist imiquimod (IMQ)., Methods: Female BALB/c mice (7-9 week old) were randomly assigned to two experimental groups: an untreated control group and a group treated topically with IMQ (IMQ-treated) for 4 or 8 weeks. A group of IMQ-treated mice that take a combination of the antioxidants tempol and apocynin, and another treated IL-17-neutralizing antibody were also performed., Results: TLR7 activation gradually increased blood pressure, associated with elevated plasma levels of anti-dsDNA autoantibodies, splenomegaly, hepatomegaly, and severe expansion of splenic immune cells with an imbalance between proinflammatory T cells and regulatory T cells. TLR7 activation induced a marked vascular remodeling in mesenteric arteries characterized by an increased media--lumen ratio (≈40%), and an impaired endothelium-dependent vasorelaxation in aortas from wild-type mice after 8 weeks of treatment. In addition, an increased ROS production, as a result of the upregulation of NADPH oxidase subunits, and an enhanced vascular inflammation were found in aortas from IMQ-treated mice. These functional and structural vascular alterations induced by IMQ were improved by antioxidant treatment. Anti-IL-17 treatment reduced blood pressure and improved endothelial dysfunction in IMQ-treated mice., Conclusion: Our results demonstrate that TLR7 activation induces the development of hypertension and vascular damage in BALB/c mice, and further underscore the increased vascular inflammation and oxidative stress, mediated in part by IL-17, as key factors contributing to cardiovascular complications in this TLR7-driven lupus autoimmunity model.

Age-related vascular endothelial dysfunction is a major antecedent to cardiovascular diseases. We investigated whether increased circulating levels of the gut microbiome-generated metabolite trimethylamine-N-oxide induces endothelial dysfunction with aging. In healthy humans, plasma trimethylamine-N-oxide was higher in middle-aged/older (64±7 years) versus young (22±2 years) adults (6.5±0.7 versus 1.6±0.2 µmol/L) and inversely related to brachial artery flow-mediated dilation ( r 2 =0.29, P <0.00001). In young mice, 6 months of dietary supplementation with trimethylamine-N-oxide induced an aging-like impairment in carotid artery endothelium-dependent dilation to acetylcholine versus control feeding (peak dilation: 79±3% versus 95±3%, P <0.01). This impairment was accompanied by increased vascular nitrotyrosine, a marker of oxidative stress, and reversed by the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl. Trimethylamine-N-oxide supplementation also reduced activation of endothelial nitric oxide synthase and impaired nitric oxide-mediated dilation, as assessed with the nitric oxide synthase inhibitor L-NAME (N G -nitro-L-arginine methyl ester). Acute incubation of carotid arteries with trimethylamine-N-oxide recapitulated these events. Next, treatment with 3,3-dimethyl-1-butanol for 8 to 10 weeks to suppress trimethylamine-N-oxide selectively improved endothelium-dependent dilation in old mice to young levels (peak: 90±2%) by normalizing vascular superoxide production, restoring nitric oxide-mediated dilation, and ameliorating superoxide-related suppression of endothelium-dependent dilation. Lastly, among healthy middle-aged/older adults, higher plasma trimethylamine-N-oxide was associated with greater nitrotyrosine abundance in biopsied endothelial cells, and infusion of the antioxidant ascorbic acid restored flow-mediated dilation to young levels, indicating tonic oxidative stress-related suppression of endothelial function with higher circulating trimethylamine-N-oxide. Using multiple experimental approaches in mice and humans, we demonstrate a clear role of trimethylamine-N-oxide in promoting age-related endothelial dysfunction via oxidative stress, which may have implications for prevention of cardiovascular diseases.

N 6 -isopentenyladenosine (i 6 A) is an RNA modification found in cytokinins, which regulate plant growth/differentiation, and a subset of tRNAs, where it improves the efficiency and accuracy of translation. The installation and removal of this modification is mediated by prenyltransferases and cytokinin oxidases, and a chemical approach to selective deprenylation of i 6 A has not been developed. We show that a selected group of oxoammonium cations function as artificial deprenylases to promote highly selective deprenylation of i 6 A in nucleosides, oligonucleotides, and live cells. Importantly, other epigenetic modifications, amino acid residues, and natural products were not affected. Moreover, a significant phenotype difference in the Arabidopsis thaliana shoot and root development was observed with incubation of the cation. These results establish these small organic molecules as direct chemical regulators/artificial deprenylases of i 6 A., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The emergence of new drugs is a major feature of the treatment history of multiple myeloma (MM), which also reflects the current incurability of MM. As a unique member of cyclin dependent kinase (CDK) family, CDK5 participates in numerous tumorigenic or non‑tumorigenic processes. The aim of this study is to investigate the effects of CDK5 on the viability of MM cells and bortezomib resistance using western blotting, immunohistochemistry, transient transfection, MTT assays, cell cycle analysis, apoptosis assays and a myeloma xenograft mouse model. The present study found that MM patients with high CDK5 expression in the bone marrow do not respond well to bortezomib, have higher DS stage and worse prognosis. Genetic and pharmacological (dinaciclib) inhibition of CDK5 triggers MM cell viability inhibition. Dinaciclib induces G2/M arrest and apoptosis of MM cells. In vivo experiments with myeloma xenograft mice indicate that dinaciclib significantly reduces the volume of tumors with good tolerance. Dinaciclib combined with bortezomib exerts a synergistic anti‑myeloma activity accompanied by inhibiting the activation of the nuclear factor‑κB pathway. This study demonstrates the important role of CDK5 in the pathogenesis, viability, prognosis and resistance to bortezomib of MM, laying a solid theoretical foundation for further clinical use of CDK5 inhibitors.

Pulmonary vasoconstriction resulting from intermittent hypoxia (IH) contributes to pulmonary hypertension (pHTN) in patients with sleep apnea (SA), although the mechanisms involved remain poorly understood. Based on prior studies in patients with SA and animal models of SA, the objective of this study was to evaluate the role of PKCβ and mitochondrial reactive oxygen species (mitoROS) in mediating enhanced pulmonary vasoconstrictor reactivity after IH. We hypothesized that PKCβ mediates vasoconstriction through interaction with the scaffolding protein PICK1 (protein interacting with C kinase 1), activation of mitochondrial ATP-sensitive potassium channels (mitoK ATP ), and stimulated production of mitoROS. We further hypothesized that this signaling axis mediates enhanced vasoconstriction and pHTN after IH. Rats were exposed to IH or sham conditions (7 h/d, 4 wk). Chronic oral administration of the antioxidant Tempol or the PKCβ inhibitor LY-333531 abolished IH-induced increases in right ventricular systolic pressure and right ventricular hypertrophy. Furthermore, scavengers of O 2 - or mitoROS prevented enhanced PKCβ-dependent vasoconstrictor reactivity to endothelin-1 in pulmonary arteries from IH rats. In addition, this PKCβ/mitoROS signaling pathway could be stimulated by the PKC activator PMA in pulmonary arteries from control rats, and in both rat and human pulmonary arterial smooth muscle cells. These responses to PMA were attenuated by inhibition of mitoK ATP or PICK1. Subcellular fractionation and proximity ligation assays further demonstrated that PKCβ acutely translocates to mitochondria upon stimulation and associates with PICK1. We conclude that a PKCβ/mitoROS signaling axis contributes to enhanced vasoconstriction and pHTN after IH. Furthermore, PKCβ mediates pulmonary vasoconstriction through interaction with PICK1, activation of mitoK ATP , and subsequent mitoROS generation.

Background: Alcohol use disorder places a heavy burden on global public health systems and thus is in urgent need of improved pharmacotherapies. Previously, our group has demonstrated that 30 mg/kg of the indole alkaloid brucine significantly attenuates alcohol-drinking behavior; however, the high toxicity, poor water solubility, short half-life, and limited therapeutic window of brucine restrain its clinical application as an antialcoholism medication. We subsequently hypothesized that the oxide of brucine (brucine N-oxide) would produce a similar behavioral effect without the risk profile associated with brucine., Methods: Male Fawn-Hooded rats with high innate alcohol preference underwent 2-bottle choice procedures (Experiments 1 to 3). Experiment 1 examined the effects of 7 daily BNO injections of 0, 30, 50, or 70 mg/kg (s.c.) on voluntary alcohol consumption (n = 9/group). Experiment 2 evaluated the impact of a single dose of 0 or 70 mg/kg BNO on the increased alcohol intake induced by a 4-day alcohol deprivation (n = 8/group). Experiment 3 tested the effect of 7 daily BNO injections of 0 or 70 mg/kg (s.c.) on sucrose preference (n = 6/group). Experiment 4 measured the median lethal dose (LD50) values of BNO and brucine to compare their acute toxicity in rats. Experiment 5 tested whether BNO (0, 30, 50, and 70 mg/kg, s.c.) affected locomotor activity using an open-field paradigm (n = 8/group). Finally, Experiment 6 evaluated the possible conditioned rewarding effects of 0, 30, 50, and 70 mg/kg BNO using the conditioned place preference paradigm (n = 6/group)., Results: BNO administration dose-dependently attenuated alcohol consumption without affecting food intake, total fluid consumption, or the natural preference for a sucrose solution, with 70 mg/kg BNO reducing consumption by 22.8%. A single dose of 70 mg/kg BNO significantly inhibited the alcohol deprivation effect. The LD50 values of BNO and brucine in rats were determined to be 1,103.5 ± 177.0 mg/kg and 264.6 ± 17.7 mg/kg, respectively. Finally, BNO administration did not affect spontaneous locomotor activity or induce a place preference., Conclusions: BNO may help to control excessive alcohol use and should be considered a treatment strategy for future study and development., (© 2020 by the Research Society on Alcoholism.)

Multifunctional metal-organic frameworks (MOFs) that possess permanent porosity are promising catalysts in organic transformation. Herein, we report the construction of a hierarchical MOF functionalized with basic aliphatic amine groups and polyvinylpyrrolidone-capped platinum nanoparticles (Pt NPs). The postsynthetic covalent modification of organic ligands increases basic site density in the MOF and simultaneously introduces mesopores to create a hierarchically porous structure. The multifunctional MOF is capable of catalyzing a sequential Knoevenagel condensation-hydrogenation-intramolecular cyclization reaction. The unique selective reduction of the nitro group to intermediate hydroxylamine by Pt NPs supported on MOF followed by intramolecular cyclization with a cyano group affords an excellent yield (up to 92%) to the uncommon quinoline N -oxides over quinolines. The hierarchical MOF and polyvinylpyrrolidone capping agent on Pt NPs synergistically facilitate the enrichment of substrates and thus lead to high activity in the reduction-intramolecular cyclization reaction. The bioactivity assay indicates that the synthesized quinoline N -oxides evidently inhibit the proliferation of lung cancer cells. Our findings demonstrate the feasibility of MOF-catalyzed direct synthesis of bioactive molecules from readily available compounds under mild conditions.

Nitroxides and nitroxide-containing nanoparticles (RNP) are excellent antioxidants. However, they have relatively high reduction potentials, which make them behave like oxidants or show little activity in some antioxidant assays. We found that stable nitroxyl radicals (TEMPO and 4-amino-TEMPO) has low reactivity in the test of scavenging of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS • ). As a result, supplementation of blood plasma with nitroxides may decrease its total antioxidant capacity assayed with ABTS • . Nitroxides oxidize Fe 2+ and in this way interfere with the ferric-Xylenol Orange assay of peroxides. Nitroxides as well as RNP directly oxidize glutathione and fluorogenic probes used for estimation of reactive oxygen species (ROS) (dihydro-2'7'-dichlorofluorescein diacetate, dihydroethidine and dihydrorhodamine 123) and thus produce artefacts in assays of glutathione and ROS in cell-free and cellular systems. These results point to the necessity of careful interpretation of antioxidant assays concerning nitroxides and RNP or performed in their presence., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) is among the top four malignancies in Saudi nationals, and it is the top leukemia subtype worldwide. Resistance to available AML drugs requires the identification of new targets and agents. Hsp90 is one of the emerging important targets in AML, which has a central role in the regulation of apoptosis and cell proliferation through client proteins including the growth factor receptors and cyclin dependent kinases. The objective of the first part of this study is to investigate the putative Hsp90 inhibition activity of three novel previously synthesized quinazolines, which showed HL60 cytotoxicity and VEGFR2 and EGFR kinases inhibition activities. Using surface plasmon resonance, compound 1 (HAA 2020 ) showed better Hsp90 inhibition compared to 17-AAG, and a docking study revealed that it fits nicely into the ATPase site. The objective of the second part is to maximize the anti-leukemic activity of HAA 2020 , which was combined with each of the eleven standard inhibitors. The best resulting synergistic effect in HL60 cells was with the pan cyclin-dependent kinases (CDK) inhibitor dinaciclib, using an MTT assay. Furthermore, the inhibiting effect of the Hsp90α gene by the combination of HAA 2020 and dinaciclib was associated with increased caspase-7 and TNF-α, leading to apoptosis in HL60 cells. In addition, the combination upregulated p27 simultaneously with the inhibition of cyclinD3 and CDK2, leading to abolished HL60 proliferation and survival. The actions of HAA 2020 propagated the apoptotic and cell cycle control properties of dinaciclib, showing the importance of co-targeting Hsp90 and CDK, which could lead to the better management of leukemia.

Tempol (4-hydroxy-2,2,6,6-Tetramethylpiperidine-1-oxyl, TPL), a nitroxide compound, inhibits proliferation and increases the vulnerability of cancer cells to apoptosis induced by cytotoxic agents. However, the molecular mechanism of TPL inhibiting cancer cell proliferation has not been fully understood. In this study, we evaluated the metabolic effect of TPL on cancer cells and explored its cancer therapeutic potential. Extracellular flow assays showed that TPL inhibited cellular basal and maximal oxygen consumption rates of mitochondrial. 13 C metabolic flux analysis showed that TPL treatment had minimal effect on glycolysis. However, we found that TPL inhibits glutamine metabolism by interfering with the oxidative tricarboxylic acid cycle (TCA) process and reductive glutamine process. We found that the inhibitory effect of TPL on metabolism occurs mainly on the step from citrate to α-ketoglutarate or vice versa. We also found that activity of isocitrate dehydrogenase IDH1 and IDH2, the key enzymes in TCA, were inhibited by TPL treatment. In xenograft mouse model, TPL treatment reduced tumor growth by inhibiting cellular proliferation of xenograft tumors. Thus, we provided a mechanism of TPL inhibiting cancer cell proliferation by interfering with glutamine utilization that is important for survival and proliferation of cancer cells. The study may help the development of a therapeutic strategy of TPL combined with other anticancer medicines.

Spinal cord injury is one of the most serious traumatic diseases. The current available clinical therapies are unable to provide effective recovery of nerve functions. Implantation of biomaterial scaffolds is a promising approach to bridge the damaged nerve tissue in the absence of the extracellular matrix. However, the treatments have been impaired by the increased generation of reactive oxygen species in the microenvironment of acute spinal cord injury. Efficient delivery of antioxidants and biocompatible materials and reagents has been a challenge. Herein, a novel hyaluronic acid (HA) hydrogel functionalized with the antioxidant compound 2,2,6,6-tetramethylpiperidinyloxy (TEMPO) is fabricated for nerve tissue regeneration after serious spinal cord transection in rats. TEMPO is tethered onto HA chains to form HA-TEMPO through a Schiff base reaction between 4-amino-TEMPO and aldehyde modified HA chains. The TEMPO-hydrogel is constructed with a highly porous three-dimensional structure via the gelation between the residue aldehydes in HA-TEMPO and the amines in adipic dihydrazide modified HA. The functional TEMPO-hydrogel exhibits the antioxidant effect in an H2O2 simulated in vitro peroxidative microenvironment. Implantation of the functional hydrogel in vivo induces a significant motor function restoration, which could be attributed to the effective functions of the TEMPO-hydrogel in tissue reconnection as well as nerve fiber regeneration of the central nervous spinal cord tissue. Importantly, the treatment with the TEMPO-hydrogel effectively protects the bladder tissue from neurogenic damage. Therefore, the functional TEMPO-hydrogel provides a promising strategy for the treatment of central nervous system diseases through the antioxidant and lesion-bridging regulation of the pathological microenvironment.

In 2016 one person died and others had neurological sequelae during a clinical trial with BIA 10-2474 (3-(1-(cyclohexyl(methyl)carbamoyl)-lH-imidazol-4-yl)pyridine 1-oxide), a novel fatty acid amide hydrolase (FAAH) inhibitor being developed for the treatment of medical conditions such as pain. Prior to the clinical trial a full battery of regulatory toxicology tests were carried out and this paper describes the genotoxicity/mutagenicity tests undertaken with BIA 10-2474 using the Ames (Salmonella typhimurium) reverse mutation test, the Escherichia coli WP2uvrA forward mutation test, an in vitro chromosome damage assay in human lymphocytes, and an in vivo micronucleus test in mice. All tests were conducted with and without a rat liver S9 metabolic activation system. None of the test results were judged to be positive with regards to the mutagenicity/genotoxicity of BIA 10-2474 making it unlikely that any such effect was involved in the toxicity observed in the clinic., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. The study was funded by Bial Portela & Companhia S.A. (São Mamede do Coronado, Portugal). Stephen B. Harris, Jerry F. Hardisty, A. Wallace Hayes, and Klaus Weber were paid consultants of Bial. Furthermore, one of the authors (KW) was the study pathologist on all of the studies. The manuscript was written by the authors and reviewed by Bial; however, the work product and conclusions are those of the authors., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The present paper describes the regulatory safety pharmacology studies that were carried out to support the clinical trial application for BIA 10-2474. Animal studies complied with worldwide regulatory guidelines (e.g. EEC Council Directive 75/318/EEC and subsequent amendments). Oral administration of BIA 10-2474 at doses of 30, 100 and 300 mg/kg to male Han Wistar rats did not cause any significant physiological or behavioral changes, affect the respiration rate or the tidal volume, the gastrointestinal transit, urinary output volume or pH, nor urinary sodium, potassium or chloride excretion. BIA 10-2474, at 30 μg/mL, slightly, but significantly, reduced the hERG outward tail currents by 10.62%, but had no effect at 1, 3 or 10 μg/mL. BIA 10-2474 (1.5, 4.5 and 15 μg/mL) had no substantial effects on resting membrane potential (RMP), maximal upstroke velocity (V max ), action potential amplitude (APA), action potential duration at 30%, 60% and 90% or action potential triangulation over the 30-min superfusion period in the dog isolated Purkinje fiber. In conscious dogs monitored by telemetry, BIA 10-2474 (20, 50 and 100 mg/kg p.o.) did not significantly modify arterial blood pressure as compared with controls (inter-group comparison), although a tendency toward an increase in arterial blood pressure was observed at 100 mg/kg, with systolic blood pressure being the most affected parameter. In conclusion, BIA 10-2474 had no adverse effects on any of the major vital organ systems, and, unfortunately, the data shed no further light on the mechanism(s) responsible for the clinical trial accident with BIA 10-2474., Competing Interests: Declaration of Competing Interest These studies were funded by Bial-Portela & Cª S.A. (São Mamede do Coronado, Portugal). Patrício Soares-da-Silva is an employee of Bial Portela & Companhia S. A. Paul Moser was previously an employee of Bial Portela & Companhia S. A. and is now acting as a paid consultant to them. A. Wallace Hayes, Peter Pressman are paid consultants of Bial-Portela & Cª S.A., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)