Usnic Acid Targets 14-3-3 Proteins and Suppresses Cancer Progression by Blocking Substrate Interaction.

The anticancer therapeutic effects of usnic acid (UA), a lichen secondary metabolite, have been demonstrated in vitro and in vivo. However, the mechanism underlying the anticancer effect of UA remains to be clarified. In this study, the target protein of UA was identified using a UA-linker-Affi-Gel molecule, which showed that UA binds to the 14-3-3 protein. UA binds to 14-3-3, causing the degradation of proteasomal and autophagosomal proteins. The interaction of UA with 14-3-3 isoforms modulated cell invasion, cell cycle progression, aerobic glycolysis, mitochondrial biogenesis, and the Akt/mTOR, JNK, STAT3, NF-κB, and AP-1 signaling pathways in colorectal cancer. A peptide inhibitor of 14-3-3 blocked or regressed the activity of UA and inhibited its effects. The results suggest that UA binds to 14-3-3 isoforms and suppresses cancer progression by affecting 14-3-3 targets and phosphorylated proteins.

2-Amino-5-arylethynyl-thiophen-3-yl-(phenyl)methanones as A1 Adenosine Receptor Positive Allosteric Modulators.

A1 adenosine receptor (A1AR) agonists have cerebroprotective, cardioprotective, antinociceptive, and other pharmaceutical applications. We explored the structure-activity relationship of 5-arylethynyl aminothiophenes as A1AR positive allosteric modulators (PAMs). The derivatives were compared in binding and functional assays at the human A1AR, indicating that some fluoro-substituted analogues have enhanced PAM activity. We identified substitution of the terminal phenyl ring in 12 (2-F-Ph), 15 (3,4-F2-Ph, MRS7935), and 21 (2-CF3-Ph) as particularly enhancing the PAM activity. 15 was also shown to act as an A1 ago-PAM with EC50 ≈ 2 µM, without activity (30 µM) at other ARs. Molecular modeling indicated that both the 5-arylethynyl and the 4-neopentyl groups are located in a region outside the receptor transmembrane helix bundle that is in contact with the phospholipid bilayer, consistent with the preference for nonpolar substitution of the aryl moiety. Although they are hydrophobic, these PAMs could provide potential drug candidate molecules for engaging protective A1ARs.

Association of pre-left ventricular assist device defibrillator shocks for ventricular arrhythmia with clinical outcomes after left ventricular assist device implantation.

Background: Implantable cardioverter-defibrillation (ICD) shocks after left ventricular assist device therapy (LVAD) are associated with adverse clinical outcomes. Little is known about the association of pre-LVAD ICD shocks on post-LVAD clinical outcomes and whether LVAD therapy affects the prevalence of ICD shocks. Objectives: The purpose of this study was to determine whether pre-LVAD ICD shocks are associated with adverse clinical outcomes post-LVAD and to compare the prevalence of ICD shocks before and after LVAD therapy. Methods: Patients 18 years or older with continuous-flow LVADs and ICDs were retrospectively identified within the University of Pittsburgh Medical Center system from 2006-2020. We analyzed the association between appropriate ICD shocks within 1 year pre-LVAD with a primary composite outcome of death, stroke, and pump thrombosis and secondary outcomes of post-LVAD ICD shocks and ICD shock hospitalizations. Results: Among 309 individuals, average age was 57 ± 12 years, 87% were male, 80% had ischemic cardiomyopathy, and 42% were bridge to transplantation. Seventy-one patients (23%) experienced pre-LVAD shocks, and 69 (22%) experienced post-LVAD shocks. The overall prevalence of shocks pre-LVAD and post-LVAD were not different. Pre-LVAD ICD shocks were not associated with the composite outcome. Pre-LVAD ICD shocks were found to predict post-LVAD shocks (hazard ratio [HR] 5.7; 95% confidence interval [CI] 3.42-9.48; P <.0001) and hospitalizations related to ICD shocks from ventricular arrhythmia (HR 10.34; 95% CI 4.1-25.7; P <.0001). Conclusion: Pre-LVAD ICD shocks predicted post-LVAD ICD shocks and hospitalizations but were not associated with the composite outcome of death, pump thrombosis, or stroke at 1 year. The prevalence of appropriate ICD shocks was similar before and after LVAD implantation in the entire cohort.

Assay-Dependent Inverse Agonism at the A3 Adenosine Receptor: When Neutral Is Not Neutral.

The A3 adenosine receptor (A3AR) is implicated in a variety of (patho)physiological conditions. While most research has focused on agonists and antagonists, inverse agonism at A3AR has been scarcely studied. Therefore, this study aimed at exploring inverse agonism, using two previously engineered cell lines (hA3ARLgBiT-SmBiTßarr2 and hA3ARLgBiT-SmBiTminiGαi), both employing the NanoBiT technology. The previously established inverse agonist PSB-10 showed a decrease in basal signal in the ß-arrestin 2 (ßarr2) but not the miniGαi recruitment assay, indicative of inverse agonism in the former assay. Control experiments confirmed the specificity and reversibility of this observation. Evaluation of a set of presumed neutral antagonists (MRS7907, MRS7799, XAC, and MRS1220) revealed that all displayed concentration-dependent signal decreases when tested in the A3AR-ßarr2 recruitment assay, yielding EC50 and Emax values for inverse agonism. Conversely, in the miniGαi recruitment assay, no signal decreases were observed. To assess whether this observation was caused by the inability of the ligands to induce inverse agonism in the G protein pathway, or rather by a limitation inherent to the employed A3AR-miniGαi recruitment assay, a GloSensor cAMP assay was performed. The outcome of the latter also suggests inverse agonism by the presumed neutral antagonists in this latter assay. These findings emphasize the importance of prior characterization of ligands in the relevant test system. Moreover, it showed the suitability of the NanoBiT ßarr2 recruitment and the GloSensor cAMP assays to capture inverse agonism at the A3AR, as opposed to the NanoBiT miniGαi recruitment assay.

Biomass waste as an alternative source of carbon and silicon-based absorbents for CO2 capturing application.

The production of low-cost solid adsorbents for carbon dioxide (CO2) capture has gained massive consideration. Biomass wastes are preferred as precursors for synthesis of CO2 solid adsorbents, due to their high CO2 adsorption efficiency, and ease of scalable low-cost production. This review particularly focuses on waste biomass-derived adsorbents with their CO2 adsorption performances. Specifically, studies related to carbon (biochar and activated carbon) and silicon (silicates and geopolymers)-based adsorbents were summarized. The impact of experimental parameters including nature of biomass, synthesis route, carbonization temperature and type of activation methods on the CO2 adsorption capacities of biomass-derived pure carbon and silicon-based adsorbents were evaluated. The development of various enhancement strategies on biomass-derived adsorbents for CO2 capture and their responsible factors that impact adsorbent's CO2 capture proficiency were also reviewed. The possible CO2 adsorption mechanisms on the adsorbent's surface were highlighted. The challenges and research gaps identified in this research area have also been emphasized, which will help as further research prospects.

Characteristics of Premature Myocardial Infarction Among Women With Prior Adverse Pregnancy Outcomes.

BACKGROUND: Adverse pregnancy outcomes (APOs), hypertensive disorders of pregnancy, gestational diabetes mellitus, and preterm birth are associated with ischemic heart disease in later life. OBJECTIVES: The authors aimed to study the features of premature myocardial infarction (MI) among women with and without prior APOs. METHODS: We performed a retrospective analysis of women with premature MI (<65 years of age) referred for left heart catheterization matched with a database of abstracted pregnancy data. We compared MI characteristics and epicardial coronary anatomy between women with and without APOs during their index pregnancy and evaluated time from delivery to MI. RESULTS: Of 391 women with premature MI and associated coronary angiography (age: 49 ± 8 years), 154 (39%) had a prior APO (hypertensive disorders of pregnancy n = 78, preeclampsia n = 35, gestational diabetes mellitus n = 28, and preterm birth n = 48). Women with APO history had a higher prevalence of diabetes (33% vs 16% without APO; P = 0.001) and presented earlier with MI following delivery (19.6 [IQR: 14.3-23.5] years vs those without APO 21.5 [IQR: 17.0-25.4] years; P = 0.012), driven by preeclampsia (17.1 [IQR: 12.7-22.4] years, P = 0.010). Women with and without APOs had similar MI features including rates of ST-segment elevation MI, obstructive and multi-vessel coronary artery disease, percutaneous coronary intervention, and shock. CONCLUSIONS: Among women with premature MIs, 39% had a history of an APO. Women with APO history presented sooner after pregnancy but had similar MI characteristics vs those without APOs. Pregnancy history may identify women who warrant early, aggressive cardiovascular disease prevention.

A2A adenosine receptor agonists, antagonists, inverse agonists and partial agonists.

The Gs-coupled A2A adenosine receptor (A2AAR) has been explored extensively as a pharmaceutical target, which has led to numerous clinical trials. However, only one selective A2AAR agonist (regadenoson, Lexiscan) and one selective A2AAR antagonist (istradefylline, Nouriast) have been approved by the FDA, as a pharmacological agent for myocardial perfusion imaging (MPI) and as a cotherapy for Parkinson's disease (PD), respectively. Adenosine is widely used in MPI, as Adenoscan. Despite numerous unsuccessful clinical trials, medicinal chemical activity around A2AAR ligands has accelerated recently, particularly through structure-based drug design. New drug-like A2AAR antagonists for PD and cancer immunotherapy have been identified, and many clinical trials have ensued. For example, imaradenant (AZD4635), a compound that was designed computationally, based on A2AAR X-ray structures and biophysical mapping. Mixed A2AAR/A2BAR antagonists are also hopeful for cancer treatment. A2AAR antagonists may also have potential as neuroprotective agents for treatment of Alzheimer's disease.

Modified poly(vinylidene fluoride) nanomembranes for dye removal from water - A review.

Water contamination due to soluble synthetic dyes has serious concerns. Membrane-based wastewater treatments are emerging as a preferred choice for removing dyes from water. Poly(vinylidene fluoride) (PVDF)-based nanomembranes have gained much popularity due to their favorable features. This review explores the application of PVDF-based nanomembranes in synthetic dye removal through various treatments. Different fabrication methods to obtain high performance PVDF-based nanomembranes were discussed under surface coating and blending methods. Studies related to use of PVDF-based nanomembranes in adsorption, filtration, catalysis (oxidant activation, ozonation, Fenton process and photocatalysis) and membrane distillation have been elaborately discussed. Nanomaterials including metal compounds, metals, (synthetic/bio)polymers, metal organic frameworks, carbon materials and their composites were incorporated in PVDF membrane to enhance its performance. The advantages and limitations of incorporating nanomaterials in PVDF-based membranes have been highlighted. The influence of nanomaterials on the surface features, mechanical strength, hydrophilicity, crystallinity and catalytic ability of PVDF membrane was discussed. The conclusion of this literature review was given along with future research.

Topical Delivery of Atraric Acid Derived from Stereocaulon japonicum with Enhanced Skin Permeation and Hair Regrowth Activity for Androgenic Alopecia.

Atraric acid (AA) is a phenolic compound isolated from Stereocaulon japonicum that has demonstrated anti-androgen properties and was used to design an alternative formulation for the treatment of alopecia. This new topical formulation was designed using a solvent mixture system composed of ethanol as a volatile vehicle, oleic acid as a permeation enhancer, and water for skin hydration. The ideal topical AA formulation (AA-TF#15) exhibited an 8.77-fold higher human skin flux and a 570% increase in dermal drug deposition, compared to 1% (w/w) AA in ethanol. In addition, compared to other formulations, AA-TF#15 (1% [w/w] AA) activated keratinocytes and human dermal papilla cell proliferation at a concentration of 50 µM AA, which is equivalent to 50 µM minoxidil. Moreover, AA-TF#15 treatment produced a significant increase in hair regrowth by 58.0% and 41.9% compared to the 1% (w/w) minoxidil and oral finasteride (1 mg/kg)-treated mice. In addition, AA-TF#15 showed a higher expression level of aldehyde dehydrogenase 1, ß-catenin, cyclin D1, and pyruvate kinase M2 proteins in the skin of AA-TF#15-treated mice compared to that of those treated with minoxidil and oral finasteride. These findings suggest AA-TF#15 is an effective formulation for the treatment of scalp androgenic alopecia.

Route to extreme events in a parametrically driven position-dependent nonlinear oscillator.

We explore the dynamics of a damped and driven Mathews-Lakshmanan oscillator type model with position-dependent mass term and report two distinct bifurcation routes to the advent of sudden, intermittent large-amplitude chaotic oscillations in the system. We characterize these infrequent and recurrent large oscillations as extreme events (EE) when they are significantly greater than the pre-defined threshold height. In the first bifurcation route, the system exhibits a bifurcation from quasiperiodic (QP) attractor to chaotic attractor via strange non-chaotic (SNA) attractor as a function of damping parameter. In the second route, the chaotic attractor in the form of EE has emerged directly from the QP attractor. Hence, to the best of our knowledge, this is the first study to report the birth of EE from these two distinct bifurcation routes. We also discuss that EE are emerged due to the sudden expansion of the chaotic attractor via interior crisis in the system. Regions of different dynamical states are distinguished using the Lyapunov exponent spectrum. Further, SNA and QP dynamics are determined using the singular spectrum analysis and 0-1 test. The region of EE is characterized using the threshold height.

Facile synthesis of CuO/g-C3N4 nanolayer composites with superior catalytic reductive degradation behavior.

The cupric oxide (CuO) loaded graphitic carbon nitride (g-C3N4) nanocomposites (CuO/g-C3N4) were prepared by a facile calcination method. The formation of monoclinic CuO nanocrystals along with g-C3N4 was confirmed by X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopic analysis. X-ray photoelectron spectral (XPS) analysis further confirms the formation of CuO/g-C3N4. Distribution of CuO stone-like crystalline nanoparticles on g-C3N4 nanosheets was observed by transmission electron microscopic images. The influence of CuO loading on the optical property of g-C3N4 was determined by ultraviolet (UV)-visible absorption and photoluminescence (PL) spectral analysis. Band gap was decreased from 2.7 to 2.3 eV by the addition of CuO nanoparticles. The catalytic performance of the synthesized samples in 4-nitrophenol (4-NP) and methyl orange (MO) reduction was evaluated. The 5 wt% CuO/g-C3N4 showed 99.5% (7 min) and 99.7% (4 min) reduction efficiency for 4-NP and MO respectively. The 5 wt% CuO/g-C3N4 could become a potential catalyst in the chemical treatment of organic pollutants.

Saroglitazar suppresses the hepatocellular carcinoma induced by intraperitoneal injection of diethylnitrosamine in C57BL/6 mice fed on choline deficient, l-amino acid- defined, high-fat diet.

BACKGROUND: Saroglitazar is a novel PPAR-α/γ agonist with predominant PPAR-α activity. In various preclinical models, saroglitazar has been shown to prevent & reverse symptoms of NASH. In view of these observations, and the fact that NASH is a progressive disease leading to HCC, we hypothesized that saroglitazar may prevent the development of HCC in rodents. METHODS: HCC was induced in C57BL/6 mice by a single intraperitoneal injection of 25 mg/kg diethylnitrosamine (DEN) at the age of 4 weeks and then feeding the animal a choline-deficient, L-amino acid- defined, high-fat diet (CDAHFD) for the entire study duration. Eight weeks after initiation of CDAHFD, saroglitazar (1 and 3 mg/kg) treatment was started and continued for another 27 weeks. RESULTS: Saroglitazar treatment significantly reduced the liver injury markers (serum ALT and AST), reversed hepatic steatosis and decreased the levels of pro-inflammatory cytokines like TNF-α in liver. It also resulted in a marked increase in serum adiponectin and osteopontin levels. All disease control animals showed hepatic tumors, which was absent in saroglitazar (3 mg/kg)- treatment group indicating 100% prevention of hepatic tumorigenesis. This is the first study demonstrating a potent PPARα agonist causing suppression of liver tumors in rodents, perhaps due to a strong anti-NASH activity of Saroglitazar that overrides its rodent-specific peroxisome proliferation activity. CONCLUSION: The data reveals potential of saroglitazar for chemoprevention of hepatocellular carcinoma in patients with NAFLD/NASH.

Anaerobic Degradation of Naphthalene and Pyrene by Sulfate-Reducing Cultures Enriched from Former Manufactured Gas Plant Soil.

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) under completely anaerobic sulfate-reducing conditions is an energetically challenging process. To date, anaerobic degradations of only two-ringed naphthalene and three-ringed phenanthrene by sediment-free and enriched sulfate-reducing bacteria have been reported. In this study, sulfate-reducing enrichment cultures capable of degrading naphthalene and four-ringed PAH, pyrene, were enriched from a contaminated former gas plant site soil. Bacterial community composition analysis revealed that a naphthalene-degrading enrichment culture, MMNap, was dominated (84.90%) by a Gram-positive endospore-forming member of the genus Desulfotomaculum with minor contribution (8.60%) from a member of Clostridium. The pyrene-degrading enrichment, MMPyr, was dominated (97.40%) by a species of Desulfotomaculum. The sequences representing the Desulfotomaculum phylotypes shared 98.80% similarity to each other. After 150 days of incubation, MMNap degraded 195 µM naphthalene with simultaneous reduction of sulfate and accumulation of sulfide. Similarly, MMPyr degraded 114 µM pyrene during 180 days of incubation with nearly stochiometric sulfate consumption and sulfide accumulation. In both cases, the addition of sulfate reduction inhibitor, molybdate (20 mM), resulted in complete cessation of the substrate utilization and sulfate reduction that clearly indicated the major role of the sulfate-reducing Desulfotomaculum in biodegradation of the two PAHs. This study is the first report on anaerobic pyrene degradation by a matrix-free, strictly anaerobic, and sulfate-reducing enrichment culture.

Fluorescent A2A and A3 adenosine receptor antagonists as flow cytometry probes.

Adenosine receptor (AR) ligands are being developed for metabolic, cardiovascular, neurological, and inflammatory diseases and cancer. The ease of drug discovery is contingent on the availability of pharmacological tools. Fluorescent antagonist ligands for the human A2A and A3ARs were synthesized using two validated pharmacophores, 1,3-dipropyl-8-phenylxanthine and triazolo[1,5-c]quinazolin-5-yl)amine, which were coupled to eight reporter fluorophores: AlexaFluor, JaneliaFluor (JF), cyanine, and near infrared (NIR) dyes. The conjugates were first screened using radioligand binding in HEK293 cells expressing one of the three AR subtypes. The highest affinities at A2AAR were Ki 144-316 nM for 10, 12, and 19, and at A3AR affinity of Ki 21.6 nM for 19. Specific binding of JF646 conjugate MRS7774 12 to the HEK293 cell surface A2AAR was imaged using confocal microscopy. Compound 19 MRS7535, a triazolo[1,5-c]quinazolin-5-yl)amine containing a Sulfo-Cy7 NIR dye, was suitable for A3AR characterization in whole cells by flow cytometry (Kd 11.8 nM), and its bitopic interaction mode with an A3AR homology model was predicted. Given its affinity and selectivity (11-fold vs. A2AAR, ~ 50-fold vs. A1AR and A2BAR) and a good specific-to-nonspecific binding ratio, 19 could be useful for live cell or potentially a diagnostic in vivo NIR imaging tool and/or therapy targeting the A3AR.

Progressive yield of nickel cobaltite nanocubes for visible light utilization and degrading activities of methyl orange dye pollutant.

In this study, pure cobalt oxide (Co3O4) as well as nickel cobaltite (NiCo2O4) were investigated with their capacity of degradation efficiency for textile dyes like methyl orange (MO) employing visible light irradiation. Two variable concentrations of nickel cobaltite (NiCo2O4) with 75:25 and 50:50 wt ratios along with the pure metal oxides were synthesized by thermal decomposition method and analyzed by various sophisticated instruments. Initially, the structural characteristics described the fine crystalline nature of NiCo2O4 and also exhibits reduced size than the pure component material (Co3O4). Besides, NiCo2O4 catalysts represented nano cubic shaped particles, and also their coordinating functional groups were evaluated. Further, the absorption wavelength confirms the two band positions of NiCo2O4 which leads to promote visible light absorption, and degrading efficiency of about 47.5% for NiCo2O4 (75:25) sample compared with NiCo2O4 (50:50) which produced only 26.3% degradation. This higher efficiency of the former was due to high crystallinity and interfacial charge transfer of combined Ni2+, Ni3+, Co2+ and Co3+ redox couples. This consecutively produces effective OH radicals that brought the degradation effectively under visible light. The recycling capacity up to 5 repeated cycles has been studied with the NiCo2O4 (75:25) and therefore the catalyst can further be used in other dye degradation.

A Case Report of Forehead Subgaleal Lipoma: Diagnostic Dilemmas and Surgical Solutions.

Subgaleal lipomas are an uncommon type of benign soft tissue tumour. They typically present as painless, slow-growing masses in the subcutaneous plane beneath the galea aponeurotica of the scalp. This case report presents a rare subgaleal lipoma in a 50-year-old female patient with a chief complaint of painful swelling on her forehead above the left eyebrow. Diagnostic imaging revealed a well-defined, round, fat-density mass in the subcutaneous plane of the bilateral frontal and high parietal midline region, causing a mass effect. The patient underwent successful excision of the subgaleal lipoma under local anaesthesia, leading to relief from the swelling and associated pain with no reported complications during the recovery period. Follow-up assessments confirmed the absence of recurrence.

Structure-Activity Studies of 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives as A3 Adenosine Receptor Positive Allosteric Modulators.

We previously reported 1H-imidazo[4,5-c]quinolin-4-amines as A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs). A3AR agonists, but not PAMs, are in clinical trials for inflammatory diseases and liver conditions. We synthesized new analogues to distinguish 2-cyclopropyl antagonist 17 (orthosteric interaction demonstrated by binding and predicted computationally) from PAMs (derivatives with large 2-alkyl/cycloalkyl/bicycloalkyl groups). We predicted PAM binding at a hydrophobic site on the A3AR cytosolic interface. Although having low Caco-2 permeability and high plasma protein binding, hydrophobic 2-cyclohept-4-enyl-N-3,4-dichlorophenyl, MRS7788 18, and 2-heptan-4-yl-N-4-iodophenyl, MRS8054 39, derivatives were orally bioavailable in rat. 2-Heptan-4-yl-N-3,4-dichlorophenyl 14 and 2-cyclononyl-N-3,4-dichlorophenyl 20 derivatives and 39 greatly enhanced Cl-IB-MECA-stimulated [35S]GTPγS binding Emax, with only 12b trending toward decreasing the agonist EC50. A feasible route for radio-iodination at the p-position of a 4-phenylamino substituent suggests a potential radioligand for allosteric site binding. Herein, we advanced an allosteric approach to developing A3AR-activating drugs that are potentially event- and site-specific in action.

Electrochemical detection of hydrogen peroxide using micro and nanoporous CeO2 catalysts.

In this research work, focus has been made on a glassy carbon electrode (GCE) modified commercial micro and synthesized nano-CeO2 for the detection of hydrogen peroxide (H2O2). Firstly, CeO2 nanoleaves were prepared by solvothermal route. Both commercially available micro CeO2 and synthesized nano-CeO2 structures were analyzed by different characterization techniques. The Raman spectra of synthesized nano CeO2 has more oxygen vacancies than micro CeO2. SEM images revealed that the synthesized CeO2 acquired leaf-like morphology. The catalyst nano CeO2 offered mesoporosity from nitrogen adsorption-desorption isotherms with massive sites of activation for increasing efficiency. Experiments on determining H2O2 using micro CeO2 or nano-CeO2/GCE was conducted using cyclic voltammetry (CV) and amperometry. Enhanced H2O2 reduction peak current with lower potential was observed in nano-CeO2/GCE. The influence of scan rate and H2O2 concentration on the performance of nano-CeO2/GCE were also studied. The obtained results have indicated that nano-CeO2/GCE showed improved electrochemical sensing behavior towards the reduction of H2O2 than micro-CeO2/GCE and bare GCE. A linear relationship was obtained over 0.001 µM-0.125 µM concentration of H2O2, with good sensitivity 141.96 µA µM-1 and low detection limit of 0.4 nM. Hence, the present nano-CeO2 system will have a great potential with solvothermal synthesis approach in the development of electrochemical sensors.

Nef inhibits HIV transcription and gene expression in astrocytes and HIV transmission from astrocytes to CD4+ T cells.

HIV infects astrocytes in a restricted manner but leads to abundant expression of Nef, a major viral factor for HIV replication and disease progression. However, the roles of Nef in HIV gene expression and replication in astrocytes and viral transfer from astrocytes to CD4+ T cells remain largely unclear. In this study, we attempted to address these issues by transfecting human primary astrocytes with HIV molecular clones with intact Nef and without Nef (a nonsense Nef mutant) and comparing gene expression and replication in astrocytes and viral transfer from astrocytes to CD4+ T cells MT4. First, we found that lack of Nef expression led to increased extracellular virus production from astrocytes and intracellular viral protein and RNA expression in astrocytes. Using a HIV LTR-driven luciferase reporter gene assay, we showed that ectopic Nef expression alone inhibited the HIV LTR promoter activity in astrocytes. Consistent with the previously established function of Nef, we showed that the infectivity of HIV derived from astrocytes with Nef expression was significantly higher than that with no Nef expression. Next, we performed the co-culture assay to determine HIV transfer from astrocytes transfected to MT4. We showed that lack of Nef expression led to significant increase in HIV transfer from astrocytes to MT4 using two HIV clones. We also used Nef-null HIV complemented with Nef in trans in the co-culture assay and demonstrated that Nef expression led to significantly decreased HIV transfer from astrocytes to MT4. Taken together, these findings support a negative role of Nef in HIV replication and pathogenesis in astrocytes.

Characterization of Dual-Acting A3 Adenosine Receptor Positive Allosteric Modulators That Preferentially Enhance Adenosine-Induced Gαi3 and GαoA Isoprotein Activation.

The A3 adenosine receptor (A3AR) is a promising therapeutic target for inflammatory diseases, cancer, and chronic neuropathic pain, with agonists already in advanced clinical trials. Here we report an in-depth comparison of the pharmacological properties and structure-activity relationships of existing and expanded compound libraries of 2-substituted 1H-imidazo[4,5-c]quinolin-4-amine and 4-amino-substituted quinoline derivatives that function as A3AR positive allosteric modulators (PAMs). We also show that our lead compound from each series enhances adenosine-induced A3AR signaling preferentially toward activation of Gαi3 and GαoA isoproteins, which are coexpressed with the A3AR in immune cells and spinal cord neurons. Finally, utilizing an extracellular/intracellular chimeric A3AR approach composed of sequences from a responding (human) and a nonresponding (mouse) species, we provide evidence in support of the idea that the imidazoquinolin-4-amine class of PAMs variably interacts dually with the orthosteric ligand binding site as well as with a separate allosteric site located within the inner/intracellular regions of the receptor. This study has advanced both structural and pharmacological understanding of these two classes of A3AR PAMs, which includes leads for future pharmaceutical development.