In the current study, we attempted to identify the critical domains or amino acid (aa) residue(s) that promote defects in HIV(IN:M50I/V151I), using a series of variants, including truncated or aa-substituted RNase H (RH) or IN. The results demonstrated that virus release and the initiation of autoprocessing were regulated by the C-terminal domains (CTDs) of RH and IN. Further studies illustrated that Asp at codon 109 of RH CTD and Asp at the C terminus of IN induces the defect. This result indicated that the CTDs of RH and IN in GagPol and particular aa positions in RH and IN regulated the virus release and the initiation of autoprocessing, and these sites could be potential targets for the development of new therapies.Previously, we reported that an HIV-1 variant containing Met-to-Ile change at codon 50 and Val-to-Ile mutation at codon 151 of integrase (IN), HIV(IN:M50I/V151I), was an impaired virus. Despite the mutations being in IN, the virus release was significantly suppressed ( Nascent viral particles of human immunodeficiency virus type-1 (HIV-1) released from HIV-1-producing cells are immature and non-infectious . The parcis) [The nascent immature HIV-1 particles mature into infectious viruses through a sequential processing step of the enzymatic cleavage of Gag and GagPol proteins by the mature PR ,3. The ncis) ; this stcis) ,13,14. Tcis) . The fircis) ,13,14. Tcis) ,16 or recis) ,18,19,20cis) ,22 or RHNL4.3, as a parent strain, and site-directed mutagenesis was conducted to induce individual or a combination of the SNPs. We found that a mutant containing a Met-to-Ile change at codon 50 of IN (IN:M50I) was impaired. The mutant suppressed the virus release to 0.3% of wild-type HIVNL4.3 (WT) and inhibited the GagPol autoprocessing step. These results indicated that IN regulates virus release and autoprocessing. Other groups reported that IN regulated maturation/processing using IN-deleted/truncated HIV mutants [A genome-wide association study of HIV genomic RNA demonstrated that a total of 14 non-synonymous single nucleotide polymorphisms (SNPs) were correlated with plasma viral load (VL) in anti-retrovirus treatment-naive HIV-infected patients . The mut mutants ,18,19,21 mutants . Interes mutants . These rpol region. We then found that the recombinant HIV(IN:M50I) virus contained a Val-to-Ile mutation at codon 151 of IN (IN:V151I) in the backbone [NL4.3 [NL4.3 IN protein-containing M50I and V151I is functional [We investigated a population of HIV variants containing the IN:M50I mutation in patients participating in NIAID clinical trials. Surprisingly, the plasma VL level in patients infected with HIV containing IN:M50I without the compensatory mutations (either IN:S17N or RH:N79S) was 1207–201,353 copies/mL , suggestbackbone . IN:V151e [NL4.3 , and is e [NL4.3 ,28,29,30nctional , while oIn the current study, we further investigated the mechanism of the defects in virus release and autoprocessing in HIV(M50I/V151I) and reported that the C-terminal domains of RH and IN are associated with the deficiency. These results provide a potential target for the development of anti-HIV drugs/therapy.Approval for these studies, including all sample materials and protocols, was granted by the National Institute of Allergy and Infectious Diseases (NIAID) Institutional Review Board, and participants were provided the informed written consent prior to blood being drawn. All experimental procedures in these studies were approved by the National Cancer Institute at Frederick and Frederick National Laboratory for Cancer Research .v/v) fetal bovine serum , and 50 μg/mL gentamicin (Thermo Fisher), as previously described [Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors’ apheresis packs (NIH blood bank) using a lymphocyte separation medium  ,24; CD4(escribed .E. coli (Thermo Fisher). The plasmid purification was carried out using the EndoFree Plasmid Maxi Kit .Plasmids encoding HIV variants were constructed by site-directed mutagenesis on pNL4.3  the pla, as prevw/v) sucrose (MiliporeSigma) in 10 mM HEPES-150 mM NaCl buffer (pH 7.4), as previously described [Recombinant HIV-1 variants were prepared by transfecting the pNL4.3 mutants into HEK293T cells using TransIT-293  and Opti-MEM I medium (Thermo Fisher) following a method previously reported . Cultureescribed ,34. The escribed ,34.v/v) FBS, and 50 μg/mL gentamicin (RP10). The PHA-stimulated CD4(+) T cells (10 × 106 cells) were infected with 10 ng of p24 of each HIV variant at 10 × 106 cells/mL in RP10 for two hours at 37 °C. The infected cells were washed with RP10, and then cultured at 1 × 106 cells/mL in the medium in the presence of 20 units/mL of recombinant IL-2 (MiliporeSigma) for 14 days at 37 °C in T25 flasks [The levels of HIV replication of each variant were determined using primary CD4(+) T cells as previously described . CD4(+) 5 flasks ,24. HalfTo confirm the proteolytic processing of Gag and GagPol polyproteins in HIV virions, Western blot (WB) analysis was performed as previously described ,24. Due https://www.rcsb.org, accessed on 29 August 2022) into PyMOL . Chain A in the structure was colored in lime (the RH part was colored in cyan), and chain B was colored in olive. RH:N79N and RH:D109 were only visible in chain A (Chain A:D630), colored in red and blue, respectively. The distance of the alpha carbon between RH:N79 and RH:D109 was calculated using the following command: distance chain A and i. 600 and n. CA, chain A and i. 630 and n. CA.The HIV Cryo-EM structure of the PR-RT structure (PDB accession #: 7sjx)  was downt-test using GraphPad Prism 9 . p-values less than 0.05 were considered statistically significant .Intergroup comparisons were performed by one-way analysis of variance (ANOVA) with multiple comparison analysis or Student’s 2+ or Mg2+) [We reported that RH:N79S or IN:S17N restored the inhibition of virus release and autoprocessing of HIV(IN:M50I/V151I) . In the or Mg2+)  requiredor Mg2+) .+ 0.02% (n = 5) (p < 0.0001) compared to HIV WT (+ 0.1% (n = 4) (p > 0.05) of HIV(WT) in the presence of RH:N79S. The GagPol processing was also rescued /region(s) of RH may be directly associated with the recovery. We decided to determine how the RH domain(s) in the M50I/V151I mutant influenced autoprocessing and the virus maturation and constructed a series of HIV(M50I/V151I) variants containing different lengths of RH B. To evao HIV WT C and res rescued A,B. All  rescued . The pro rescued A,B. The http://www.hiv.lanl.gov, accessed on 13 July 2022), Stanford drug-resistance database , and NIAID HIV sequence data. The population analysis indicated that RH:D109 was highly conserved among the databases  of HIV(WT) (The defect of HIV(IN:M50I/V151I) in the virus-release process was partially rescued in the absence of RH C, implic HIV(WT) A, indica HIV(WT) B,C; thusAs shown above, RH:D109N rescued the defects in autoprocessing and virus release in the context of HIV(IN:M50I/V151I). This implicated that the residue or the α4 helix domain of RH negatively regulated both the virus release and the initiation of autoprocessing in the context. To further understand the suppression mechanism in the presence of RH:D109 and the role of domains of IN in inducing the defect, we constructed a series of variants A. IN is p < 0.05, n = 4)  and autoprocessing (The virus release of the mutant lacking the entire IN (ΔIN) was suppressed to 49.7 ± 8.5% of that of WT  B, and th, n = 4) C,D. Therocessing C,D. Of iocessing B–D, in cWe also assessed the capability of HIV replication and compared its activity among the mutants. It is reported that variants lacking the last 15~17 aa residues at the tail domain diminished enzymatic function in IN and infectivity ,41; thisWe considered how the tail restored the autoprocessing, even in the presence of RH:D109. Crystal structural analysis of the full length of IN has not yet been successful because of the flexibility and instability of the tail, and its poor solubility under low salt conditions ,42,43,44p < 0.05)  is relatively conserved in HIV sequences  ; thus, t < 0.05) A; howeve < 0.05) B,C, and  < 0.05) D, indicaTo confirm the function of the last codon, we created another mutant lacking the codon in the setting of context, HIV(IN:M50I/V151/Δ288). Intriguingly, the virus lacking the last amino acid of IN could fully recover virus release and autoprocessing activities A. HoweveAs described in the Introduction section, we had previously reported that the RH:N79S or IN:S17N mutation rescued the defect of HIV(IN:M50I/V151I) replication . In the We have previously demonstrated that a combination of M50I and V151I mutations in IN suppresses virus release by accumulating the abnormal size of buds on the surface of HIV-producing cells. In the presence of the combination, the processing of Gag and GagPol was inhibited at the initiation of autoprocessing ,24. RH:NA series of HIV(IN:M50I/V15I) variants containing aa substitutions at IN:D288 illustrated that six aa changes  could play a role as compensatory mutations for virus release, autoprocessing, and virus fitness of HIV(IN:M50I/V151I) without the previously described rescue mutations. In order to define the clinical settings, we conducted a population analysis using NIAID HIV sequences and the LANL HIV database. We previously reported that 17 sequences in LANL (14 subtype B and 3 subtype C) contained IN:M50I/V151I mutations without the rescue mutations . We expeThe results of WB from HIV(ΔIN) and HIV(IN:M50I/V151/ΔCTD) demonstrated that both mutants contained less PR than HIV(WT). By contrast, HIV(IN:M50I/V151I/Δ14aa) and HIV(IN:M50I/V151I/D288G) carried a comparable amount of PR to HIV(WT) . The banThe sequential deletion of aa residues from the C-terminal of IN(WT) revealed that the IN C-terminal tail region (aa residues 270–288) is required for HIV-1 infection, reverse transcription, and maturation ,20. A fuAutoprocessing is initiated at the junction between p2 and NC in intramolecular mechanisms . In our p < 0.05, n = 5). In addition, a mutant lacking codon 288, HIV(IN:M50I/V151I/Δ288), was able to restore virus release and autoprocessing, but virus replication was 10~20% of HIV(WT) on day 7. Therefore, the virus release, autoprocessing, and virus replication may be independently regulated in a C-terminal aa-dependent manner.Using RH-truncated mutants in HIV(IN:WT) and HIV(IN:M50I/V151I), our study demonstrated that the RH domain is involved in virus release; ΔRH suppressed HIV(IN:WT) release, but ΔRH partially rescued HIV(IN:M50I/V151I) release. In contrast, the WB results demonstrated that the amounts of Gag/GagPol processing in both ΔRH mutants were comparable to that of HIV(WT). It appears that the RH domain is not involved in regulating the initiation of autoprocessing. IN:D288G/K/E/Y fully restored virus release, autoprocessing, and viral replication. The IN:D288N change also nearly fully recovered the processing, however, it partially rescued the virus release of HIV(IN:M50I/V151I) to 48.6 ± 6.3% of that of HIV(WT)  was defective in virus release and autoprocessing followed by maturation; thus, the mutant was replication-incompetent ,24. The


The conjugation of a dopamine-binding aptamer (DBA) to the pA-AuNPsyields aptananozyme structures catalyzing simultaneously the H2O2-mediated oxidation of dopamine to aminochromethrough the aerobic oxidation of glucose. A set of aptananozymes consistingof DBA conjugated through the 5′- or 3′-end directlyor spacer bridges to pA-AuNPs were synthesized. The set of aptananozymesrevealed enhanced catalytic activities toward the H2O2-catalyzed oxidation of dopamine to dopachrome, as comparedto the separated pA-AuNPs and DBA constituents, and structure–functionrelationships within the series of aptananozymes were demonstrated.The enhanced catalytic function of the aptananozymes was attributedto the concentration of the dopamine at the catalytic interfaces bymeans of aptamer–dopamine complexes. The dual catalytic activitiesof aptananozymes were further applied to design bioreactors catalyzingthe effective aerobic oxidation of dopamine in the presence of glucose.Mechanistic studies demonstrated that the aptananozymes generate reactiveoxygen species. Accordingly, the AS1411 aptamer, recognizing the nucleolinreceptor associated with cancer cells, was conjugated to the pA-AuNPs,yielding a nanozyme for the chemodynamic treatment of cancer cells.The AS1411 aptamer targets the aptananozyme to the cancer cells andfacilitates the selective permeation of the nanozyme into the cells.Selective cytotoxicity toward MDA-MB-231 breast cancer cells (ca. 70% cell death) as compared to MCF-10A epithelial cells(ca. 2% cell death) is demonstrated.Polyadenine-stabilized Au nanoparticles (pA-AuNPs) revealdualnanozyme catalytic activities toward the H Organiccatalytic particles, including, for example, melamine23 or polydopamine24 nanoparticles,and NMOFs, such as Zr-based NMOFs modified with metal ions26 or MOF-818,27 have revealed nanozymeactivities. Diverse enzyme activities have been emulated by nanozymes,including peroxidase,30 oxidase,33 laccase,34 superoxide dismutase,35 catalase,36 isomerase,37 and hydrolase39 activities. Also, hybrid multi-enzyme nanoparticle carriers or hybridsconsisting of enzyme/nanozyme nanoparticles have been reported ascomposite nanoreactors for operating catalytic or biocatalytic cascades.41 Different applications of nanozymes43 or nanoreactorcomposites44 have been demonstrated, includingtheir use as sensors46 and imaging agents,48 in medical applications such as cancer therapy,50 for treatment of diseases such as Alzheimer’s52 or Parkinson’s disease,54 and for catalyticrelease of cardiovascular drugs.55 In addition,nanozymes have been used as antibacterial and wound-healing agentsby generating reactive oxygen species (ROS)56 and applied as catalysts for the degradation of pollutants.57Substantial research efforts are directedto the development ofinorganic, organic, or metal–organic framework nanoparticles(NMOFs) as catalysts mimicking native enzymes, 2+-ion-modified C-dots nanozyme with β-cyclodextrinreceptor binding units or the coating of nanocatalysts with molecularlyimprinted matrices.58While nanozymes demonstrate enhanced stabilitiesas compared tonative enzymes, they reveal lower catalytic activities and lack thestereoselectivity and chiroselectivity that are fundamental featuresof enzymes. As the high catalytic stereoselectivity and chiroselectivityfunctions of native enzymes originate from the well-defined structuraland chiral architecture of the active site that provides cooperativebinding of the substrate  and spatial proximityof the substrate to the catalytic site, efforts to mimic these featuresby nanozymes have been reported. These include the functionalizationof the Cu61 or catalyticproperties of nucleic acids (DNAzymes)62 have been recently applied to develop nanozymes.64 The conjugation of aptamers to DNAzymes yielded enzyme-like conjugates(nucleoapzymes) revealing improved binding of the reaction substrateand enhanced catalytic and chiroselective properties.66 In addition, the functionalization of Cu2+-ion-modifiedC-dots with the dopamine aptamer or the tyrosinamide aptamer yieldedhighly active nanozymes for the oxidation of dopamine to aminochrome,the chiroselective oxidation of l-/d-DOPA to l-/d-dopachrome, and oxygen insertion into aryl C–Hbonds of tyrosinamide for its oxidation into amidodopachrome. Thisclass of aptamer-modified nanozymes was termed by us “aptananozymes”.67The sequence-specificrecognition properties of nucleic acids (aptamers)68 such as sensing,69 imaging,70 assemblyof chiroplasmonic structures71 and switches,72 construction of optical nanodevices,73 and the use of their thermoplasmonic propertiesas nanothermometers.74 Recently, polyadenine-stabilizedAuNPs (pA-AuNPs) demonstrated peroxidase-like nanozyme activities75 reflected by the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS2–) by H2O2 toform ABTS•–.Nucleic acid-modified gold nanoparticles(AuNPs) have found diverseapplications,2O2. Furthermore, we find that the pA-AuNPs act,also, as a nanozyme that catalyzes the aerobic oxidation of glucoseto gluconic acid and H2O2. This allows the useof pA-AuNPs as a bioreactor nanozyme that catalyzes the cascaded aerobicoxidation of dopamine to aminochrome in the presence of glucose. Thatis, in contrast to native enzymes that reveal a dictated catalyticfunction, we find that a single AuNP nanozyme can catalyze two differentcatalytic transformations, e.g., catalyzed oxidationof dopamine by H2O2 and aerobic oxidation ofglucose. This not only demonstrates the diversity of nanozymes vs. native enzymes, but also allows us to apply a singlenanozyme as a bioreactor that activates a cascaded biocatalytic processwhere the catalyzed aerobic oxidation of glucose to gluconic acidand H2O2 catalyzes oxidation of dopamine toaminochrome. The peroxidase activity of pA-AuNPs toward the oxidationof dopamine is, however, quite low. To enhance the nanozyme activity,we tether a series of dopamine-binding aptamers (DBAs) directly totheir 3′- or 5′-end or through spacer units to yieldaptananozyme bioreactors, 2O2 and a 13-fold-enhancedoxidation of dopamine in the presence of glucose, as compared to theseparated nanozyme/aptamer units. In addition, we demonstrate thechiroselective oxidation of l-/d-DOPA to l-/d-dopachrome by H2O2 and the DBA-functionalizedpA-AuNPs, aptananozyme, and the chiroselective oxidation of l-/d-DOPA to l-/d-dopachrome by glucoseand the aptananozyme bioreactor. It should be noted that previousstudies demonstrated the catalytic activities of “naked”AuNPs toward the aerobic oxidation of glucose,31 yet surface functionalization of the AuNPs led to catalyticallyinactive particles. Thus, the demonstration that pA-AuNPs retain theirglucose oxidase nanozyme activities is significant and moreover importantas it allows the conjugation of aptamer tethers to the pA-AuNPs thatenable sequestered biocatalytic cascades by the aptananozyme reactorand aptamer-guided therapeutic applications (vide infra) by the aptananozyme. The mechanism of the DBA-functionalized pA-AuNPs-catalyzedoxidation of dopamine by H2O2 involves the generationof •OH as ROS intermediates.Here we wish to reporton the peroxidase-like nanozyme activityof pA-AuNPs toward the oxidation of dopamine to aminochrome by H•OH by thepA-AuNPs is used to engineer AS1411 aptamer-functionalized pA-AuNPsas functional aptananozymes for the chemodynamic treatment of MDA-MB-231cancer cells. The cytotoxicity of the AS1411/pA-AuNPs toward MDA-MB-231cancer cells and toward MDA-MB-231 xenogragt tumor-bearing mice isaddressed.The glucose-mediated generation of 75Supporting Information, Figures S1–S3.)The resulting pA-AuNPs reveal peroxidase-like activities and resultin the oxidation of dopamine by H2O2 to formaminochrome, Figure S4. In addition, wefind that pA-AuNPs reveal glucose oxidase activities and, in the presenceof glucose, catalyze the aerobic oxidation of glucose to gluconicacid and H2O2, Figure S5. The dual catalytic activities of the pA-AuNPs allowed, then, theuse of the pA-AuNPs as a nanozyme bioreactor for the catalyzed aerobicoxidation of dopamine to aminochrome in the presence of glucose, Figure S6. In this system, the aerobic oxidationof glucose to gluconic acid and H2O2, and theresulting H2O2 in the vicinity of the catalyticparticles, allows the subsequent cascade oxidation of dopamine toaminochrome. The pA-AuNPs were synthesizedaccording to the reported procedure,2O2-catalyzed oxidation of dopamine toaminochrome and the cascaded aerobic oxidation of dopamine, in thepresence of glucose, is moderate, as compared to other peroxidase-mimickingnanozymes. The importance of the systems rests on the dual catalyticbioreactor activities of the particles. To improve the catalytic activitiesof the pA-AuNPs system, we applied the concept of aptananozyme, wherethe DBA was tethered to the pA-AuNPs. In these systems, the DBA tetheredto the particles concentrate the dopamine substrate at the aptamer-bindingsite, thereby allowing the effective utilization of H2O2, While the pA-AuNPs reveal dual catalyticactivities and establisha nanozyme bioreactor function, the effectiveness of the system towardthe H1)to the pA stabilizer units (aptananozyme I) and the 5′-endaptamer sequence (2) to the pA sequence (aptananozymeII), and the 5′-end aptamer tethered to the pA sequence via a four-base spacer TGTA bridge (3), aneight-base spacer (TGTA)2 (4), and a 12-basespacer (TGTA)3 sequence (5) . (The aptamer and aptamer–spacersequences elongated at the respective 3′- or 5′-endof the pA hairpin were used to synthesize the pA-AuNPs.) All pA-AuNPsinclude an identical loading of the modified pA-aptamer strands onthe AuNPs . 2O2) usingthe aptananozyme I, curve (a), aptananozyme II, curve (b), aptananozymeIII, curve (c), and aptananozyme IV and V, curves (d) and (e), respectively.The time-dependent absorbance changes corresponding to aminochromeformation were applied to follow the kinetics of dopamine oxidationby the respective aptananozymes. For comparison, the rate of oxidationof dopamine by H2O2 in the presence of the pA-AuNPsmodified with the nucleic acid (2a), consisting of thescrambled bases of DBA, is presented in curve (f), and the rate ofoxidation of the dopamine by the separated pA-AuNPs/aptamer constituentsin the presence of H2O2 is displayed in curve(g). The catalytic rates of the aptamer-modified nanozymes reveala Michaelis–Menten-type kinetic behavior and reach saturationlevels, consistent with the saturation of the aptamer-binding receptorsites. All aptamer-modified nanozymes show substantially enhancedcatalytic oxidation of dopamine, as compared to the control systemsof the separated nanozyme/aptamer couple or the nanozyme conjugatedby scrambled bases aptamer (2a). The enhancement of thedopamine oxidation rate by the aptamer-modified nanozymes followsthe order I < II < III < IV, indicating the following: (i)The tethering of the 5′-end to the pA-AuNPs yields a nanozymeof slightly higher catalytic activity as compared to the 3′-end-modifiedaptamer tethered to the pA-AuNPs. (ii) The introduction of a spacerunit bridging the 5′-end of the aptamer to the pA-AuNPs improvesthe catalytic performance of the nanozyme, and the eight-bases-bridgedspacer leads to superior catalytic properties of the nanozyme IV >III. Nonetheless, further elongation of the spacer bridge to 12 basesdecreases the catalytic performance of the nanozyme V < IV. Thecatalytic properties of the aptamer-functionalized pA-AuNPs nanozymesfollow the binding properties between the aptamer–pA-AuNPsconjugates and dopamine substrate. (iii) The kinetic features, KM and Vmax, associatedwith the different aptamers-functionalized pA-AuNPs are summarizedin Kd) of different aptananozymesto dopamine substrates are also included in Figure S7 and (4)-modified pA-AuNPs reveal a 10-fold enhanced activity as comparedto the separated aptamer/pA-AuNPs constituents. The enhanced catalyticactivities of the aptananozymes are attributed to the binding of dopamineto the aptamer units resulting in the local concentration of the reactionsubstrate (molarity effect) at the catalytic interface. That is, theaffinity binding of dopamine to the aptamer receptor controls thecatalytic efficacies of the aptananozymes, similar to the functionsof native enzymes, where the affinity binding of the substrates guidesthe biocatalytic performance. (ii) The order of the catalytic activitiesfollows the order of disassociation constants to the aptamer units.The introduction of the oligonucleotide-TGTA units improves the bindingfeatures of the aptamer toward dopamine. Presumably, the AuNPs surfaceperturbs the association of dopamine to the aptamer-binding sites,and the spacer units added to the aptamer scaffolds introduce flexibilityinto the aptamer sequence that eliminates the steric binding perturbationtoward the ligand introduced by the particles. (iii) For the long12 (TGTA)3 bridge, a decrease in the catalytic activityis observed. This is attributed to the spatial separation of the substratefrom the catalytic interface. Similar steric effects of long spacerbridges were observed for other nucleoapzyme66 and aptananozyme systems.67 (iv) Therandom sequence aptamer-modified pA-AuNPs, curve (v), reveal slightlyenhanced oxidation of dopamine, as compared to the separated pA-AuNPs/aptamerconstituents. This slight rate enhancement is attributed to the electrostaticconcentration of the positively charged dopamine at the catalyticinterface. A series of DBAs were conjugated to thepA units stabilizing theAuNPs, Figure S8, and the accompanying discussion depictsthe glucose oxidase activities of the aptananozyme IV. In this experiment,the aerobic oxidation of glucose yields gluconic acid and H2O2, and the resulting H2O2 is quantitativelyprobed by the H2O2 oxidation of Amplex Red tothe fluorescent Resorufin. All DBA-functionalized pA-AuNPs, i.e.,aptananozymes I–V, reveal similar glucose oxidase activities,and their activities are very similar to the oxidase activity of thepA-AuNPs lacking the conjugated DBA aptamer. That is, the glucoseoxidase activities of the pA-AuNPs are not influenced by the coupledDBA aptamer sequences, Figure S9. Realizingthat the series of DBA-conjugated pA-AuNPs, aptananozymes I–V,exhibit dual nanozyme catalytic functions toward the aerobic oxidationof glucose and peroxidase activities toward the oxidation of dopamineby H2O2 to aminochrome, the set of aptananozymeswas applied as bioreactor systems to guide the cascaded catalyticoxidation of dopamine by the aerobic oxidation of glucose, The DBA-functionalized pA-AuNPs, aptananozymes, revealoxidase-likecatalytic activities toward the oxidation of glucose, 2O2. For comparison, the ratesof oxidation of dopamine in the presence of the (2a) nucleicacid-modified pA-AuNPs, where (2a) includes the scrambledsequence of the DBA conjugated to the pA sequence-stabilized AuNPs,and the oxidation rates of dopamine by the separated pA-stabilizedAuNPs and the DBA, in the presence of glucose, are presented in curves(f) and (g), respectively. All aptananozyme bioreactors stimulatethe glucose-driven aerobic catalytic cascade, leading to the formationof aminochrome. While the cascaded oxidation of dopamine in the presenceof glucose and the bare pA-AuNPs is inefficient, curve (g), effectiveglucose-driven oxidation of dopamine in the presence of all aptananozymeassemblies proceeds, following the order aptananozyme I < aptananozymeII < aptananozyme III < aptananozyme IV. The aptananozyme V,consisting of the 12 spacers bridges separating the dopamine aptamerfrom pA stabilizing AuNPs, reveals the lowest cascaded activity amongthe aptananozyme assemblies. The aptananozyme IV, consisting of the8 spacers separating the dopamine aptamer from pA stabilizing theAuNPs, reveals a ca. 13-fold enhanced cascade activityas compared to the bare pA-AuNPs and the separated DBA, curve (d) vs. curve (g). Interestingly, the rates of aerobic oxidationof glucose by the (4)-pA-AuNPs and the non-aptamer-conjugatedpA-AuNPs (in the presence of the separated DBA) to form gluconic acidand H2O2 are almost similar, Figure S9. Thus, effective oxidation of dopamine by the (4)-stabilized pA-AuNPs  can beattributed to the dopamine aptamer linked to the aptananozyme bioreactorassembly. That is, for the separated pA-AuNPs/DBA system, the concentrationof dopamine near the nanozyme interface is low. As a result, the H2O2 formed by the aerobic oxidation of glucose escapesto the bulk solution, leading to inefficient diffusional oxidationof dopamine. In turn, the concentration of dopamine by the aptananozymecatalyst tethered with aptamer unit (4) (or other aptamerstructures) leads to effective utilization of the H2O2 generated by the aptananozyme-catalyzed aerobic oxidationof glucose, resulting in the efficient cascaded oxidation of dopamine,forming aminochrome. That is, the formation of H2O2 at the DBA-functionalized pA-AuNPs interface yields a highlocal concentration of H2O2 for the sequestered,cascaded oxidation of dopamine.44  The efficiencies of the catalyzedglucose-driven aerobic oxidation of dopamine to aminochrome by aptananozymesfollow the order I < II < III < IV and fit well with the Kd values of different aptananozymes toward dopamineand their efficacies to concentrate dopamine at the bioreactor interface.l-DOPA or d-DOPA upon binding tothe chiral aptamer would yield aptamer–ligand complexes ofdifferent binding affinities. Accordingly, the concentration of l-DOPA or d-DOPA at the (4)-modified pA-AuNPswill be dictated by the affinities of diastereomeric ligand–aptamercomplexes, and thus, chiroselective oxidation of l-/d-DOPA should proceed. l-DOPA, curve (a),and d-DOPA, curve (b), by H2O2, 20mM, in the presence of (4)-functionalized pA-AuNPs. Forcomparison, curves (c) and (d) show the rates of oxidation of l-/d-DOPA by the non-aptamer pA-stabilized AuNPs. The(4)-modified pA-AuNPs, aptananozyme IV, reveals chiroselectiveoxidation of the DOPA ligands, and the rates of oxidation of l-DOPA are ca. 2.1-fold enhanced as compared to therates of oxidation of d-DOPA, 4)-aptamer-binding site (Kd of l-DOPA and d-DOPA to the (4)-modified pA-AuNPs corresponding to 4.2 μM and 11.3 μM,respectively, Figure S11). That is, thehigher binding affinity of l-DOPA to the aptamer site improvesthe concentration of the ligand at the catalytic interface, resultingin the chiroselective oxidation. In turn, the polyA-non-aptamer-modifiedAuNPs do not show any chiral discrimination toward the oxidation of l-/d-DOPA, l-/d-DOPA by thechiral interface are too weak to induce any detectable chiroselectiveoxidation. The chiroselective oxidation ofl-/d-DOPAby H2O2 to dopachrome was, then, extended toinclude the chiroselective oxidation of l-/d-DOPAby applying the pA-AuNPs-DBA aptananozyme bioreactor system to catalyzethe aerobic oxidation of glucose as the H2O2 source. l-/d-DOPA by the glucose and aptananozyme bioreactor in the presenceof variable concentrations of l-/d- DOPA. The oxidationof l-DOPA, curve (a), is two-fold enhanced as compared tothe rate of oxidation of d-DOPA, curve (b). For comparison,the rates of oxidation of the l-/d-DOPA, at differentconcentrations of l-/d-DOPA, by the separated pA-AuNPsnanozyme reactor and the diffusional dopamine aptamer are displayedin curves (c) and (d). The rates of l-/d-DOPA oxidationby the separated glucose/nanozyme reactor are very low, and no chiroselectivityis detectable. The enhanced oxidation rates of l-/d-oxidation are attributed to concentration of the l-/d-DOPA at the catalytic aptananozymes reactor interface by meansof the aptamer constituents. The chiroselectivity follows the enhancedbinding affinity of l-DOPA, as compared to d-DOPA,to the chiral aptamer units.The chiral features of the dopamine aptamer suggestthat the diastereomericinteractions of 2O2 or upon interaction with glucose under aerobic conditions.Electron paramagnetic resonance (EPR) experiments confirm that subjectingthe aptananozymes to H2O2 yields hydroxyl radicals, Figure S12A, as ROS. Similarly, treatment ofthe aptananozyme with glucose under aerobic conditions leads to thedetection of the hydroxyl radical, •OH, as ROS, Figure S12B. Control experiments indicated thattreatment of the aptananozyme with glucose, under nitrogen, did notlead to hydroxyl radicals, Figure S12C.These results are consistent with the supporting experiments thatindicated the aptananozyme catalyzed aerobic oxidation of glucoseto gluconic acid and H2O2. The resulting H2O2 provides, then, the source for •OH. That is, the formation of hydroxyl radicals as the reactive speciesin the catalyzed oxidation of the catechol substrates by H2O2, or the aptananozyme-catalyzed oxidation of the catecholsubstrates in the presence of glucose, under aerobic conditions, isconfirmed. Furthermore, addition of dopamine to the mixture of H2O2 and the aptananozyme or the mixture of glucoseand the aptananozyme led to the depletion of the •OH in the systems, Figure S12D,E, implyingthat the •OH is consumed by dopamine. Accordingly,and in view of a detailed kinetic study examining the reaction of •OH with catechol derivatives,76 a possible mechanism for the •OH-driven formationof aminochrome is provided in Figure S13. Along this path, the •OH-driven oxidation ofthe catechol substrates yields a geminal ortho bis-phenoxy biradicalthat is a canonic form of the o-benzoquinone state.The intramolecular 1,4-Michael addition within the ortho quinone productsof dopamine or l-/d-DOPA yields the aminochromeor dopachrome products.An important aspect involves addressing the mechanisticpath bywhich the DBA-functionalized pA-AuNPs, i.e., the aptananozymes, catalyzethe oxidation of the different catechol derivatives by H78 The limitations associated with the use of nanozymes for chemodynamictreatment of cancer cells include, however, poor cellular permeationand insufficient selective discrimination between cancer cells andnormal cells. The discovery that pA-AuNPs catalyze the aerobic oxidationof glucose (present in cancer cells) to form gluconic acid and H2O2, while generating intermediate ROS products,suggested that the pA-AuNPs could act as reactive nanozymes for chemodynamictreatment of cancer cells. However, to overcome the difficulties associatedwith nanozymes for chemodynamic treatment of cancer cells, we decidedto conjugate the AS1411 aptamer, that binds to the nucleolin receptorassociated with different cancer cells,80 to the pA-AuNPsnanozymes as a means to target the nanozyme to cancer cells and facilitatethe permeation of the nanozymes into the cancer cells, thereby enhancingthe selectivity and chemodynamic efficacy of the nanozymes. Accordingly,the AS1411 aptamer tethered to the pA-hairpin, (6), wasused to stabilize the formation of single hybrid AS1411 aptamer–pA-AuNPs, 2O2 or upon treatment with glucose underaerobic conditions. The formation of the ROS species was confirmedby the reported assay81 that followed thedepletion of the absorbance of 1,3-diphenyl-isobenzofuran (DPBF) uponreaction with the ROS products. 2O2 and the (6)-pA-AuNPs nanozyme, and 6)-pA-AuNPs nanozyme-catalyzed oxidationof glucose under aerobic conditions. 2O2 or O2/glucose in the presence or absenceof the (6)-AuNPs.  The results indicate that the nanozymeis, indeed, essential to generate the ROS intermediates.Recent studies demonstrated that ROSgenerated by nanozymes actas cytotoxic agents against cancer cells, and thus these catalyticnanostructures may act as chemodynamic agents for cancer therapy.ex =488 nm; λem = 517 nm). N = 4 frames at each time interval). Evidently, only theMDA-MB-231 cancer cells treated with the AS1411-conjugated pA-AuNPsdemonstrate the accumulation of the ROS products, 2/glucose-driven catalyzed generation of the ROS products.The selective chemodynamic treatment of the MDA-MB-231 cancer cellsby the ROS agents is demonstrated in ca. 70% of cell death was observed.Using a series of concentrations of the AS1411-pA-AuNPs probing thecytotoxicity toward MDA-MB-231 breast cancer cells, Figure S15, the IC50 value of the aptananozymewas estimated to be ca. 1.27 nM. The results areconsistent with the selective aptamer-assisted permeation of the AS1411/pA-AuNPsinto the cancer cells.The capacity of the AS1411 aptamer/pA-AuNPs conjugateto generateROS products was, then, applied to examine the use of the NPs as intracellularagents for targeted chemodynamic treatment of cancer cells. In theseexperiments, MDA-MB-231 breast cancer cells and MCF-10A epithelialbreast cells were treated with the AS1411 aptamer/pA-AuNPs with theaim to probe the potential selective cytotoxicity of the NPs towardcancer cells that are associated with the nucleolin receptor. As aresult, the ROS generation capacity revealed by the aerobic catalyzedoxidation of intracellular glucose, invivo experiments, 3) were subjected to intra-tumoral(IT) injection of the AS1411/pA-AuNPs or control systems consistingof pA-stabilized AuNPs, lacking the aptamer tether, pA-AuNPs, or pA-stabilizedAuNPs conjugated to a randomized base sequence of the AS1411 aptamer,r-AS1411/pA-AuNPs. The tumor-bearing mice were subjected to a totalnumber of seven injections at time intervals of 2–3 days.  •OH as toxic ROS agent. Furthermore, The chemodynamic cytotoxicity of the AS1411/pA-AuNPstoward MDA-MB-231breast cancer cells was, then, followed by preliminary 2O2 that is coupled tothe effective hydrogen peroxide-catalyzed oxidation of dopamine (or l-/d- DOPA) to aminochrome (or l-/d-dopachrome). The aptamer strands linked to the nanozyme particlesprovide binding sites for the association of the catechol substatesin spatial proximity to the catalyst. Concentration of the substratesat the catalytic surface resulted in effective oxidation of the substrateand provided chiral receptor binding sites for the chiroselectiveoxidation of l-/d-DOPA to l-/d-dopachrome. The binding modes of the aptamers and the strands’flexibilities dominated by spacer bridges affect the binding propertiesof the aptamers and the resulting enhanced catalytic activities ofthe aptananozymes. An important result of the study is, however, thedemonstration that a single nanoparticle stimulates simultaneouslytwo different catalytic processes. This feature of the aptananozymeis rarely observed for native enzymes that catalyze a single targetor chemical transformation. The dual catalytic activities of the artificialaptananozyme enabled a cascaded catalytic transformation within theconfined structure of the aptananozyme that acted as a bioreactor.The mechanistic analysis of the aerobic oxidation of the catecholsubstrates by glucose demonstrated the participation of ROS intermediatesin the reactions.This study has expanded the scope of aptamer-modifiednanozymes(aptananozymes) to a system performing simultaneous and cascaded catalysis.The system involved the pA-AuNPs-catalyzed aerobic oxidation of glucoseto gluconic acid and Hin vivo experiments in MDA-MB-231 xenograft tumor-bearing mice revealedthe high chemodynamic cytotoxic efficacy of the AS1411/pA-AuNPs. Thefunctionalization of the pA-AuNPs with sequence-specific aptamers,and their capacity to yield ROS products through the aerobic oxidationof glucose, makes it possible to design functional pA-AuNPs aptananozymesfor other cancer biomarkers, diverse diseases, and antibacterial applications.The capacity of the pA-AuNPs to generate ROSproducts by the aerobicoxidation of glucose was further applied to tailor aptamer-functionalizedpA-AuNPs conjugates for the chemodynamic treatment of cancer cells.In these experiments, the AS1411 aptamer, recognizing the nucleolinreceptor associated with different cancer cells, was conjugated tothe pA-AuNPs nanozyme, to yield a functional selective nanozyme catalystfor the chemodynamic treatment of cancer cells, using the aerobicoxidation of glucose as the source for the intracellular formationof ROS products. The selective permeation and chemodynamic treatmentof MDA-MB-231 cancer cells, as compared to MCF-10A epithelial cells,was demonstrated. In addition, preliminary The sequences of the nucleic acidsused in the study are shownhere.1: 5′-CGACGCCAGTTTGAAGGTTCGTTCGCAGGTGTGGAGTGACGTCGGTAACAACAGTCAAAAAAAAAAAAAAAAAAAAGACTGTTGTTAC-3′2: 5′-GTAACAACAGTCAAAAAAAAAAAAAAAAAAAAGACTGTTGTTACCGACGCCAGTTTGAAGGTTCGTTCGCAGGTGTGGAGTGACGTCG-3′3: 5′-GTAACAACAGTCAAAAAAAAAAAAAAAAAAAAGACTGTTGTTACTGTACGACGCCAGTTTGAAGGTTCGTTCGCAGGTGTGGAGTGACGTCG-3′4: 5′-GTAACAACAGTCAAAAAAAAAAAAAAAAAAAAGACTGTTGTTACTGTATGTACGACGCCAGTTTGAAGGTTCGTTCGCAGGTGTGGAGTGACGTCG-3′5: 5′-GTAACAACAGTCAAAAAAAAAAAAAAAAAAAAGACTGTTGTTACTGTATGTATGTACGACGCCAGTTTGAAGGTTCGTTCGCAGGTGTGGAGTGACGTCG-3′6: 5′-GTAACAACAGTCAAAAAAAAAAAAAAAAAAAAGACTGTTGTTACGGTGGTGGTGGTTGTGGTGGTGGTGGTTT-3′2a: 5′-GTAACAACAGTCAAAAAAAAAAAAAAAAAAAAGACTGTTGTTACGACTAGCGTGTGTGATGGGACCTTAGGCCGTCACGGGGCTTAGT-3′DBA: 5′-CGACGCCAGTTTGAAGGTTCGTTCGCAGGTGTGGAGTGACGTCG-3′pA-strand: 5′-GTAACAACAGTCAAAAAAAAAAAAAAAAAAAAGACTGTTGTTAC-3′Supporting Information.The detailed procedure to prepare the different aptananozymes,their structural characterization, and the kinetic experiments toevaluate their catalytic functions of the aptananozymes are detailedin the


Nipah virus (NiV) and Hendra virus (HeV) are classified as high-consequence zoonotic viruses characterized by high pathogenicity and high mortality in animals and humans. Rapid diagnosis is essential to containing the outbreak. In this study, the henipavirus receptor ephrin B2 was examined to determine whether it could be used as a universal ligand for henipavirus detection in immunoassays. Enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant ephrin B2 as the capture ligand and two monoclonal antibodies (mAbs) as detection reagents. Using mAb F27NiV-34, which cross-reacts with NiV and HeV, we were able to detect NiV and HeV, while mAb F20NiV-65 was used to detect NiV. Therefore, using these two ELISAs, we were able to differentiate between NiV and HeV. Furthermore, we developed a rapid lateral flow strip test for NiV detection using ephrin B2 as the capture ligand combined with mAb F20NiV-65 as the detector. Taken together, our results show that the combination of ephrin B2 and a specific mAb provides an excellent pairing for NiV and HeV detection. Nipah virus (NiV) and Hendra virus (HeV) are classified as high-consequence zoonotic viruses and are characterized by high pathogenicity and high mortality in animals and humans ,2,3. BotHeV was first described in 1994 during an outbreak of the respiratory and neurologic disease in horses and humans in Hendra, Australia. Since its emergence, HeV continues to pose a serious public health threat to the equine and veterinary industries. HeV causes sporadic disease outbreaks in Northeastern Australia. There were around 53 incidents associated with HeV that have been reported since July 2016 ,8. NiV sParamyxoviridae, under the genus Henipavirus. Like all other viruses in the subfamily Paramyxovirinae, the genomes of NiV and HeV are non-segmented, single-stranded negative-sense RNA [NiV and HeV belong to the family ense RNA . Henipavense RNA ,13,14.Ephrin B2 and B3 have been identified as functional cellular receptors for henipaviruses, with a high affinity for the henipavirus surface glycoprotein G ,16,17. EThere is a licensed HeV vaccine for horses in Australia, however, there are currently no NiV and HeV vaccines licensed for use in humans ,21. As sg for 30 min. Virus titers were determined using virus plaque assays [6 plaque-forming units (pfu)/mL for NiV-B, 8.25 × 106 pfu/mL NiV-M, and 4.44 × 107 pfu/mL for HeV. Viruses were inactivated using 10 mM 2-Bromoethylamine Hydrobromide  [The viruses used in this study, NiV-M (Accession No. AF212302), NiV-B (Accession No. AY988601.1), and HeV (Accession No. NC_001906.3) were provided by the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia and the Public Health Agency of Canada (PHAC) in Winnipeg, Manitoba. The virus preparation procedure was described previously by Berhane et al. . Brieflye assays . The plaTrichoplusia ni (Tni) cells (Expression Systems) at a multiplicity of infection (MOI) of 5 to 10. The infected cells were harvested 72 h post-infection. Recombinant NiV-G was purified using Ni-NTA resin , and the final product was confirmed by Western blot.Full-length NiV-B glycoprotein (G) lacking the cytomembrane and transmembrane domains (GenBank: AY988601.1) was synthesized with codon optimization and cloned into pAB-bee™ –FH vector containing a polyhedron promoter  by GenScript . The purified pAB-bee™ –FH vector containing the NiV-G gene was co-transfected with linearized baculovirus vector DNA, ProFold™-ER1  into Sf9 insect cells (Expression System) to generate a recombinant baculovirus containing a NiV-G gene. The recombinant baculovirus was plaque purified. A single clone was subcultured on Sf9 cells at 27 °C with shaking at 130 rpm for 120 h and subsequently used to infect suspension cultures of Soluble form of His-tagged recombinant human ephrin B2 was expressed and purified by GenScript Inc. . Briefly, ephrin B2 (GenBank: NP_004084 (1-227)) was synthesized with codon optimization and was cloned into a pcDNA3.4 vector at the Cloning Sites of EcoRI (G/AATTC)/HindIII (A/AGCTT) and fused with His-tag at C-terminal. The purified pcDNA3.4 vector containing the ephrin B2 gene was transfected into the HD 293F cell line using a High Density (HD) Transient Expression system, the supernatant was collected, and then the His-tagged ephrin E2 protein was purified by one-step purification using an affinity column.Recombinant NiV-G and human ephrin B2 proteins were separated with 10% NuPAGE Novex Bis-Tris gels  followed by protein transfer to nitrocellulose (NC) membranes using the iBlot Gel Transfer Device . Membranes were blotted in PBS–T with 5% skim milk at room temperature for 1 h and then incubated with the following antibodies: a horseradish peroxidase (HRP)-conjugated mAb against the His-tag , a polyclonal rabbit anti-NiV-G antibody , or an anti-ephrin B2 mAb . Following primary antibody incubation, blots were incubated with an HRP–conjugated anti-rabbit or anti-mouse secondary antibody  for 1 h at room temperature. Blots were developed using the substrate 3,3′–Diaminobenzidine .Mouse immunization and fusion were performed as previously described . BrieflyMicrotitre plates  were coated with recombinant NiV-G, NiV-N  or Ebola virus glycoprotein  in a carbonate/bicarbonate buffer, pH 9.6 overnight at 4 °C. Following blocking with Casein blocking buffer , mAbs in casein blocking buffer were added. After incubation, HRP–conjugated anti-mouse IgG  was added. Then 3,3′,5,5′-Tetramethylbenzidine  was added. After stopping, optical density (OD) was measured at 450 nm using an Emax microplate reader . Each incubation step was 60 min at 37 °C with gentle shaking followed by washing five times with a washing buffer (PBS with 0.1% Tween 20).Microtitre plates  were coated with rabbit poly anti-NiV-G  or recombinant ephrin B2, or B3  in a carbonate/bicarbonate buffer at a pH 9.6 overnight at 4 °C. Following blocking with casein blocking buffer, inactivated viruses  were added to the plates, and then mAbs  or polyclonal sera (1:500)  diluted Plaque Reduction Neutralization Tests (PRNT) were carried out under CL4 containment and performed as previously described .3.Ephrin B2 biotin conjugation is performed using the BiotinTag Micro Biotinylation Kit . BAC- Sulfo N-hydroxysulfosuccinimide (NHS) was dissolved using 30 µL dimethylsulfoxide (DMSO) and then 0.1 M PBS was added to a final concentration of 5 mg/mL. The BAC-Sulfo-NHS (10 µL) was added to the recombinant human ephrin B2 (1 mg) in 0.1 M PBS and incubated for 30 min at room temperature. Following incubation, the unbound biotin was removed by extensive dialysis against PBS at 4 °C. The reagent was stored at 4 °C in PBS with 0.01% NaNMonoclonal antibody purification and gold conjugation were performed as previously described . BrieflyThe purified mAb was gold conjugated using the High Sensitivity Conjugation kit . Briefly, 70 µg 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 140 µg Sulfo-NHS were added to 1 mL Gold Nanospheres and incubated for 30 min at room temperature to activate carboxyl gold. The gold solution was washed twice with a 1 mL potassium phosphate reaction buffer (pH 7.4). The purified mAb (20 µg) was added to the gold solution and incubated for 1 h, followed by washing twice with a reaction buffer. The gold conjugated mAb was re-suspended in the conjugation buffer .The rapid assay device (gRAD) used in this study was purchased from Bioporto Diagnostics A/S . The test principle was described in a previous report . BrieflyFollowing mouse immunization with the BEI-inactivated NiV-M virus, splenocytes were harvested and fused with myeloma cells. Supernatant from the resulting hybridomas was screened using NiV-M as the antigen in an ELISA, positive clones were subcloned, and mAbs were isolated.To demonstrate mAb reactivity to NiV and/or HeV, polyclonal anti-NiV-G antibodies were coated to capture inactivated NiV and HeV. FMDV was used as a negative control in sandwich ELISA. Ideally, another paramyxovirus would be used as a negative control instead of FMDV. FMDV was used in this study as no other viruses were available in our laboratory. Two mAbs, F20NiV-65 and F27NiV-34, showed reactivity to both NiV-M and NiV-B. F27NiV-34 also reacted strongly with HeV, while F20NiV-65 failed to react with HeV in the ELISA a. Both mRecombinant NiV-G was analyzed by Western blot analysis prior to its use, which revealed a protein band with a predicted molecular weight of approximately 70 kDa . The reaThe recombinant human ephrin B2 protein was analyzed by Western blot. A band of approximately 40 kDa corresponding to the predicted molecular weight of ephrin B2 was detected using both anti-His and anti-ephrin mAbs . To deteTo confirm that ephrin B2 captures henipaviruses by interacting with NiV-G, a second ELISA was performed. Plates were coated with ephrin B2 followed by recombinant NiV-G, NiV-N, or EBOV-GP. Since only NiV-G (not NiV-N and EBOV-GP) is expected to be captured by ephrin B2, NiV-positive pig serum should only react with NiV-G. The ELISA results showed that NiV-positive pig sera detected NiV-G, but not NiV-N and EBOV-GP, while EBOV-positive sera did not detect EBOV-GP , suggestIn most antigen detection ELISAs, polyclonal antibodies are used as the capture ligand. To determine whether recombinant ephrin B2 could act similarly to polyclonal anti-NiV-G antibodies as a capture ligand in a sandwich ELISA, ephrin B2 and polyclonal anti-NiV-G were coated onto separate plates. Both inactivated NiV and HeV were detected using the mAb F27NiV-34, which reacts to both NiV and HeV. The ELISA results showed similar detection using both polyclonal NiV-G- and ephrin B2-coated plates, indicating that ephrin B2 and polyclonal anti-NiV-G antibodies have the same capture ability .Given that ephrin B2 can replace polyclonal antibodies as a ligand in ELISAs to detect henipavirus, we next sought to establish a more specific antigen detection ELISA using mAbs F27NiV-34 and F20NiV-65. Since mAb F27NiV-34 reacts with both NiV and HeV, it was used to detect both henipaviruses, while mAb F20NiV-65, which reacts only with NiV, was used to detect NiV only. Using these two ELISAs, we can differentiate between NiV and HeV. The results showed that both NiV and HeV were detected in the ELISA using mAb F27NiV-34, whereas only NiV was detected using mAb F20NiV-65 a. No rea6 pfu/mL), NiV-M (8.25 × 106 pfu/mL), HeV (4.44 × 107 pfu/mL), or FMDV were mixed with biotinylated ephrin B2 and either gold-conjugated F20NiV-65 or F27NiV-34. When the gRAD test strip is dipped into a tube containing this mixture, a positive result is indicated when the ephrin B2/virus/mAb complex reacts with the immobilized biotin-binding protein on the test line to make the test line visible. The validity of the test is confirmed when excess gold-conjugated detection mAb binds to the immobilized anti-mouse antibody to form the control line. When mAb F27NiV-34 was used as the detection mAb, no visible test line was observed in the gRAD test strip (data not shown), despite the fact that this antibody reacted to NiV and HeV-G in earlier ELISAs. In contrast, F20NiV-65, which is specific for NiV-G, produces strong bands on the test line when detecting NiV-B and NiV-M but not FMDV  testing, need to be deployed to the field. Compared to laboratory-based diagnosis, POC tests have less onerous requirements for personnel training and equipment. Therefore, they can be carried out more easily in the field or outbreak site. The LFI strip test is by far one of the most successful analytical platforms to perform the on-site detection of target substances . CurrentThe Henipavirus receptor ephrin B2 can be used as a capture ligand for NiV and HeV detection in ELISA and LFI strip tests. Ephrin B2 shows a similar ability to capture both NiV and HeV compared to polyclonal anti-NiV-G in antigen detection ELISA. This finding provides valuable information for the future development of immunoassays for accurate and rapid detection of NiV and HeV. Since the main goal of this report is to approve the concept that recombinant ephrinB2 can be used as a capture ligand for the detection of Henipaviruses, the ELISA and LFI strip test developed in this study are both prototypes.Currently, only inactivated viruses are used. In the future, if possible, both virus detection methods will be validated using a live virus, samples from experimentally infected animals, and samples from the field. The diagnostic specificity and sensitivity of ELISA and lateral flow strip tests have not been established, but they will be determined later. The sensitivity of both tests can be further improved by titration of capture and detection reagents. While neither virus detection method will be as sensitive as RT-PCR, it is hoped that the sensitivity of the strip test will be comparable to the virus detection ELISA.


EHBP1), tubulin beta class I (TUBB), and WW domain-containing oxidoreductase (WWOX) single nucleotide polymorphisms (SNPs) and coronary artery disease (CAD) and ischemic stroke (IS) are not yet understood. This study aimed to detect the associations of these SNPs, gene-gene and gene-environment interactions and CAD and IS in the Guangxi Han population. A total of 1853 unrelated subjects were recruited into normal control (n = 638), CAD (n = 622), and IS (n = 593) groups. Related genotypes were determined by high-throughput sequencing. The genotypic and minor allelic frequencies of rs2278075 were different between the CAD and control groups, and those of rs2710642, rs3130685, and rs2278075 were also different between the IS and control groups. The rs2278075T allele, rs3130685-rs2222896-rs2278075, rs3130685-rs2222896-diabetes, rs3130685-rs2222896-drinking, and haplotype rs2710642A-rs10496099C-diabetes interactions were associated with increased risk, while G-T-G-C-G-A and G-T-T-T-G-T-drinking were associated with reduced risk of CAD. The rs2278075T and rs2710642G alleles, rs2710642G-rs10496099C haplotype, rs3130685-rs2278075-rs2222896, and rs2710642-rs2278075-hypertension interactions aggravated the association with IS, whereas the rs3130685T allele, rs2710642A-rs10496099C haplotype and the interactions of H1 (s2710642A-rs10496099C)-H2 (rs2710642G-rs10496099C)-drinking and I1 (A-C-G-C-A-A)-I3 (A-C-G-T-A-A)-I4 (A-C-G-T-G-A)-I5 (G-T-G-C-G-A) diminished the association with IS. Carrying WWOX rs2278075T was strongly associated with CAD or IS, while EHBP1 rs2710642 and TUBB rs3130685 might alter the association of IS by modifying the serum lipid profile. This study demonstrates that the EHBP1, TUBB, and WWOX SNPs, gene-gene and gene-environment interactions are associated with the risk of CAD and IS in the Guangxi Han population.The associations among the EH domain-binding protein 1 ( Cardiovascular disease (CVD) including cerebrovascular disease is the leading causes of death worldwide. In 2015, the global mortality of CVD was approximately 32.1%, of which coronary artery disease (CAD) accounted for 49.8% and ischemic stroke (IS) accounted for 68% . DyslipiEHBP1) (TUBB), and WW domain-containing oxidoreductase gene (WWOX)  and Rab10. Rab10-EHBP1-EHD2 forms a trimer complex that plays a critical role in hepatocyte lipid phagocytosis and the autophagic digestion of lipid droplets (PCSK9) reduces the recycling of low-density lipoprotein receptors (LDLRs) and redirects LDLRs to late endosomes and lysosomes for degradation, facilitating an increase in serum lipids. A study found that the expression of the EHBP1 protein was downregulated in cells overexpressing PCSK9. This revealed the existence of the EHBP1 accompanying pathway linked to lipid metabolism (EHBP1 were correlated with low-density lipoprotein cholesterol (LDL-C)  A1 pathway, and it binds to a series of potential ligands of proline-rich PPxY motifs and other kinds of cell proteins. It is also involved in a variety of other pathways, including cholesterol homeostasis, fatty acid biosynthesis and triglyceride (TG) metabolism (WWOX. The correlation between the WWOX rs2222896AA genotype and LDL-C was two times that of the rs2222896AG or rs2222896 GG genotypes (WWOX rs2278075 SNP was associated with CVD (p = 5.0 × 10−9)  , tubuline (WWOX)  and dysle (WWOX) , atherosransport . EHBP1 idroplets . The protabolism . Moreove (LDL-C)  and athe (LDL-C) , respectdiseases . A genomC levels . WWOX entabolism , GWAS inenotypes . Additio × 10−9) . In a prprofiles . HoweverEHBP1, TUBB, and WWOX in the Guangxi Han population and analyzed the relationship between SNPs, gene-gene (G × G) and gene-environment (G × E) interactions, and the risk of CAD and IS, to explore the possible pathogenic mechanism of these genes and provides a theoretical basis for early CAD or IS prevention and treatment.This study investigated the genotypes of SNPs  in The subjects of our cohort were inpatients and health physical examiners in the First Affiliated Hospital of Guangxi Medical University from September 2009 to December 2011. Data were listed in the database of our study team project, including their demographic characteristics, socioeconomic status, medical history, lifestyle factors, blood samples, and laboratory results. This study was conducted in accordance with the Declaration of Helsinki (2008 revised edition) and approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University. Informed consent was obtained from all subjects.A total of 622 patients in the CAD group were diagnosed according to the following criteria: ① typical clinical manifestations, myocardial enzymes, and electrocardiogram changes; ② coronary angiography examination revealing at least one major coronary artery  stenosis ≥50%; and ③ no cerebrovascular disease, valvular heart disease, congenital heart disease, or aneurysm. A total of 593 patients in the IS group were diagnosed according to the following findings: ① large atherosclerotic stroke and/or small-artery occlusive stroke ; and ② np > 0.05). The mean age and percentage of women in the control, CAD and IS groups were 62.07 ± 12.19 years, 26.5%; 62.34 ± 10.50 years, 27.8%; and 62.85 ± 12.17 years, 27.0%, respectively. They had no evidence of liver or kidney disease, autoimmune diseases, hematological diseases, tumors, severe infections, metabolic diseases (except diabetes), or mental diseases.All participants were Han population and unrelated individuals whose ancestors lived in Guangxi for at least three generations. The ages of the subjects ranged from 18 to 80 years. Mean age and sex ratio of the three groups were matched  was collected in the morning after fasting for more than 8 h. Serum lipid parameters were determined by an automatic analyzer . Serum LDL-C, total cholesterol (TC), TG and high-density lipoprotein cholesterol (HDL-C) were measured by commercially available enzyme assay kits, and serum ApoA1 and ApoB were measured by a turbidimetric immunoassay.2 and >28 kg/m2, respectively . ③ Information on each SNP came from NCBI dbSNP Build 132 (http://www.ncbi.nlm.nih.gov/snp/). ④ SNPs with minor allele frequencies (MAFs) > 10% were included. ⑤ Associations between these SNPs  and lipid metabolism or atherosclerotic cardiovascular disease in European and American populations were identified by previous studies.The basic principles of gene and SNP selection were as follows: ① via a Shimadzu UV-1601 spectrophotometer, indicating that the DNA sample was pure. A HiSeqXTen sequencer  was employed for SNP genotyping.DNA from peripheral blood samples was extracted using the phenol-chloroform method and stored at −80°C. Approximately 10 μL (10 ng/μL) of each sample was sent to the Department for Next-Generation Sequencing, Sangon Biotech Co., Ltd.  for genotyping. The absorbance of DNA samples met a ratio of A260 nm/A280 nm = 1.8 t-test  or one-way analysis of variance (associations between three genotypes and lipid levels). Nonnormally distributed data are shown as interquartile ranges and medians, and differences in groups were determined by Mann-Whitney nonparametric tests. Differences in qualitative variables were analyzed by chi square tests. Associations between genotypes or haplotypes and continuous serum lipid levels were analyzed by multivariable linear regression. Differences in serum lipid levels associated with genotypes or haplotypes were considered statistically significant at p < 0.0004 or p < 0.0005, and G × G or G × E interactions were considered significant at p < 0.001 . The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by multiple logistic regression analysis and adjusted by stratified risk factors, including age, sex, cigarette smoking, alcohol consumption, BMI, diabetes, and hypertension. The Hardy-Weinberg equilibrium (HWE), genotypic and haplotypic frequencies, and linkage disequilibrium (LD) described by D′ and R2 values were calculated by SHEsis online software . Generalized multifactor dimensionality reduction (GMDR) online software (http://sourceforge.net/projects/gmdr/) was used to screen optimal SNP-SNP, haplotype-haplotype, G × G, and G × E interaction models. The models meet 10/10 cross-validation consistency (CV) indicating 1,000 permutation tests; both training balance accuracy and test balance accuracy were ≥50%. Furthermore, all permutation tests had p < 0.001, suggesting their accuracy predictive value of diseases. An intuitive interactive tree diagram was used to depict the association of each factor in the model, here, the factors were no more than four. All other data visualizations were generated using GraphPad Prism (version 8.0.0).Statistical analysis was performed using SPSS software version 25 . Normally distributed data are presented as the means ± standard deviation, and differences among groups were analyzed by Student’s unpaired p ≤ 0.001), whereas mean DBP, HDL-C, ApoA1, the ratio of ApoA1 to ApoB, and the percentage of subjects who consumed alcohol were lower (p ≤ 0.004) in CAD than in control groups. Mean height, SBP, DBP, pulse pressure, TG, the percentages of subjects who consumed alcohol or smoked cigarettes, and the prevalence of hypertension were higher (p ≤ 0.003), whereas mean HDL-C, ApoA1, and the ratio of ApoA1 to ApoB were lower (p ≤ 0.001) in IS than in control groups.As shown in p > 0.05 for all). The genotype and allele frequencies of the rs2278075 SNP were different between the CAD and control groups (p < 0.001 for each), and the genotype and allele frequencies of the rs2710642, rs3130685, and rs2278075 SNPs were different between the IS and control groups (p < 0.003).As listed in EHBP1, TUBB, and WWOX SNPs are shown in p = 0.0001 for the dominant model). The rs2710642GA/GG and rs2278075AT/TT genotypes were associated with increased risk of IS , whereas the rs3130685CT/TT genotypes were associated with decreased risk of IS .The genetic models of six p < 0.0004). High levels of TC and TG were associated with the rs2710642G, rs10496099T, rs3132584T, rs3130685T, and rs2222896A alleles; low levels of HDL-C were associated with the rs2710642G, rs10496099T, rs3132584T, and rs2278075T alleles. High levels of LDL-C were associated with the rs2710642G, rs10496099T, and rs3132584T alleles. Low levels of ApoA1 were associated with the rs2710642G, rs10496099T, rs3132584T, and rs3130685T alleles; low ratios of ApoA1/ApoB were associated with the rs2710642G, rs10496099T, and rs3130685T alleles .As shown in  alleles . The resEHBP1 rs2710642 and rs10496099 SNPs in the three groups . Four common haplotypes were detected in the control and disease groups (p = 7.76 × 10−9), whereas the rs2710642A-rs10496099C haplotype was associated with a reduced risk of IS . Moreover, the two haplotypes interacted with other risk factors for IS . However, there was no association between haplotypes and CAD (p > 0.05).There was strong LD between the e groups . The rs2s for IS  exhibitep < 0.025; p < 0.0005; In the control group, the levels of TG, HDL-C and ApoA1 were different between the rs2710642A-rs10496099C haplotype carriers and the haplotype noncarriers (p = 0.000), A-C-G-T-G-A , and G-T-T-T-G-T  were correlated with an increased risk of CAD. On the other hand, A-C-G-C-G-A , A-C-G-T-A-A , and G-T-G-C-G-A  were correlated with a decreased risk of CAD. Additionally, four interactions of rs2710642-rs1,0496099-rs3132584-rs3130685-rs2222896-rs2278075 were associated with IS. Namely, A-C-G-T-G-A  and G-T-G-T-G-A  were correlated with an augmented risk of IS, while A-C-G-T-A-A  and G-T-G-C-G-A  were correlated with a reduced risk of IS.As listed in p < 0.001 for all) were significantly associated with CAD  were significantly related to IS , and those drinking but not carrying G-T-T-T-G-T (I6) had lower risk of CAD than those carrying I6 but not drinking . In the IS group, compared to those carrying the rs3130685CC or rs2222896AA genotypes, subjects carrying the rs3130685CT + TT and rs2222896AG + GG genotypes were more likely to have a high risk of IS . Compared to those carrying the rs2710642GA + GG and rs2278075AA genotypes, subjects carrying the rs2710642AA and rs2278075AT + TT genotypes had an increased risk of IS . Compared to those carrying the rs2710642AA and rs2278075 TT genotypes, subjects carrying the rs2710642GA + GG and the rs2278075AT + TT genotypes had an increased risk of IS . In addition, compared to those not carrying H1 and not drinking, subjects carrying H1 and drinking had lower risk of IS , and compared to those not drinking but carrying H2, subjects drinking but not carrying H2 had lower risk of IS .Logistic regression analysis was used to further verify the association between the interactions and CAD or IS , and theThe principal findings of the present study are as follows: ① The rs2278075T allele was associated with an increased risk of CAD. The rs2278075T and rs2710642G alleles were associated with an increased risk, while the rs3130685T allele with a reduced risk of IS. ② The rs2710642G-rs10496099C haplotype was correlated with an increased risk of IS, but the rs2710642A-rs10496099C haplotype was correlated with a decreased risk of IS. ③ Serum lipid phenotypes of rs2278075T, rs2710642G and rs3130685T allele carriers were associated with dyslipidemia, and the haplotype of rs2710642A-rs10496099C was associated with beneficial lipid profiles in the control group. ④ The SNPs of rs2278075, rs2710642 and rs3130685 might be associated with CAD or IS by modifying the serum lipid profiles.WWOX were related to coronary artery calcification  between the EHBP1 rs2710642 and rs10496099 SNPs in the three groups. We found that the rs2710642A-rs10496099C haplotype reduced the risk of IS by 0.7 times, and rs2710642G-rs10496099C haplotype increased the risk of IS by 2.94 times. These results indicate that either coeffects or haplotypes of SNPs have a better predictive effect than any single SNP in the disease risk model of CAD or IS.This study found that the MAF of rs2278075 was different between the CAD and control groups, and the MAFs of rs2710642, rs3130685, and rs2278075 were different between the IS and control groups. Moreover, subjects carrying rs2278075TT had an association of a 1.58-fold risk of CAD to those carrying rs2278075AA. This was consistent with a previous GWAS in Caucasian participants , and anofication . These cngle SNP , and thengle SNP . There whttps://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/), the genotypic frequencies and MAFs of the SNPs in Han Chinese individuals in Beijing, China (CHB) were as follows: rs2278075AA 68.3%, AT 19.5%, TT 12.2%, and T 22.0%; rs2710642AA 36.6%, AG 56.1%, GG 7.3% and G 35.4%; and rs3130685CC 20.0%, CT 51.1%, TT 28.9%, and C 45.6%. It exhibited that the mutations and minor alleles of these SNPs were similar between the CHB and Guangxi Han population. Therefore, these SNPs might be associated with an aggravate risk of CAD or IS in CHB population.Previous studies reported that the rs2710642A and rs3130685T alleles were associated with LDL-C in European, Japanese and the Maonan population in China, and the rs2278075T allele was correlated with CAD in Caucasian . This stEHBP1, TUBB, and WWOX and G × E were involved in the pathogenesis of CAD and IS. The interaction of rs2710642 and rs2278075 SNPs was associated with higher risk of IS. The results also showed that the haplotype of rs2710642A-rs10496099C or rs2710642G-rs10496099C interacted with some factors  to significantly reduce the risk of IS. Some studies have shown that the level of sex hormones and complement of sex chromosomes determined sex differences in CVD patients. Many lifestyles and environmental factors  were associated with CVD in gender-specific ways. Differences in sex hormone-dependent receptors that control endothelial function may lead to increased CVD and IS risks in elderly men and postmenopausal women and a slightly lower risk of CVD and IS in premenopausal women (The interactions between al women . Our stual women . A smallal women . The resal women . In thisal women . Howeveral women . Clinicaal women . This stSome deficiencies in this study cannot be ignored. First, it did not investigate the dietary habits of the subjects in the three groups and did not analyze the effect of diet as an environmental factor. Second, we did not record the types of antihypertensive or hypoglycemic drugs taken, which cannot be ruled out because of the effect of these drugs on blood lipid levels. Third, there was no resequencing of gene expression after treatment and no follow-up of the prognosis of susceptible gene carriers in the control group. Fourth, a larger cohort sample is needed to verify whether the study has selection bias. Finally, some CAD and IS patients were taking some drugs that may affect serum lipid profiles. Therefore, we could not determine the association between the SNPs and serum lipid levels in CAD and IS groups.EHBP1, TUBB, and WWOX SNPs, G × G and G × E, and CAD and IS in the Guangxi Han population. We found that the WWOX rs2278075T allele was associated with the risk of CAD and IS, and EHBP1 rs2710642 and TUBB rs3130685 SNPs might be correlated with IS risk by regulating serum lipid profiles. The haplotypes of EHBP1 rs2710642 and rs10496099 were more predictive of IS risk than a single SNP. The interactions of G × G and G × E, such as female sex, drinking, smoking, hypertension and diabetes, may alter the association between single risk factor and CAD or IS.This study illustrated the associations among


Non-small-cell lung cancer (NSCLC) is one of the major diseases that threaten human health, and there is still no fundamental treatment method. Emerging evidences suggested that circRNAs might be an effective target to treatment NSCLC. However, the roles and detailed mechanisms of hsa_circ_0003176 in NSCLC still not clear.  hsa_circ_0003176 was identified from GSE101684 and GSE112214 datasets of Gene Expression Omnibus (GEO) database. The expression of hsa_circ_0003176 was detected by RT-qPCR in NSCLC tissues, paired adjacent nontumor tissues, and cell lines. RNA fluorescence in situ hybridization and nuclear and cytoplasmic RNA fractionation analysis was used to detect the subcellular localization of hsa_circ_0003176 in H1299 and A549 cells. Dual-luciferase reporter and RNA pull-down assay were used to confirm the regulatory of miR-182-5p to hsa_circ_0003176 and RBM5. The roles of hsa_circ_0003176 in NSCLC progression was evaluated both in vitro by CCK-8 assay, colony formation assay, wound-healing assay, and matrigel transwell assay and in vivo by the subcutaneous xenograft nude mouse experiment and lung metastasis nude mouse experiment. In addition, RNA pull down and luciferase reporter assays were carried out to investigate the interaction between hsa_circ_0003176 or RBM5 and miR-182-5p.  Our results indicated that hsa_circ_0003176 showed typical characteristic of circRNAs, which was downregulated in both NSCLC tissues and cell lines. Functionally, overexpression of hsa_circ_0003176 suppressed the proliferation, migration, and invasion of NSCLC cells in vitro and inhibited NSCLC growth and metastasis in vivo. Furthermore, we found that hsa_circ_0003176 acts as sponge of miR-182-5p to regulate RBM5 expression. Further, in vitro rescue experiments demonstrated that hsa_circ_0003176 suppressed the proliferation, migration, and invasion of NSCLC cells by regulating miR-182-5p/RBM5 axis.  We demonstrated that hsa_circ_0003176 suppressed the NSCLC progression via regulating miR-182-5p/RBM5 axis. These data indicated that hsa_circ_0003176 might be a novel molecular target for NSCLC treatment. Lung cancer is the malignant tumor, which presented the highest morbidity and mortality all over the world . Based oCircular RNAs (circRNAs) are a new class of noncoding RNAs and widely exist in the cytoplasm of eukaryotic cells . circRNAAlthough current studies have demonstrated that circRNAs play an important role in the pathogenesis of NSCLC, the biological functions and regulatory mechanisms of most circRNAs in NSCLC remain still unknown and need to be further explored. Hereby, we identified that hsa_circ_0003176 (a circRNA derived from TBX5) was significantly downregulated in NSCLC tissues compared with match adjacent nontumorous tissues through analyzing the public lung cancer dataset in the GEO database and further to explored the biological function and underlying molecular mechanism of hsa_circ_0003176 in the development of NSCLC. Our study not only disclosed a novel mechanism of NSCLC development but also provided a new molecular target for NSCLC treatment.A total of 10 NSCLC tissues and 10 corresponding adjacent nontumor tissues were collected from the University of Hong Kong-Shenzhen Hospital . These tissue specimens were reviewed and approved by the Committees for Ethical Review of Research at the University of Hong Kong-Shenzhen Hospital .https://www.ncbi.nlm.nih.gov/gds/). Receiver operating characteristic (ROC) curve assay was used to detect the area under curve (AUC) of sensitivity and specificity. circBase (http://www.circbase.org/) and circRNADb databases (http://reprod.njmu.edu.cn/cgi-bin/circrnadb/circRNADb.php) were used to investigate the localization and the transcript of hsa_circ_0003176. The potential binding miRNAs of hsa_circ_0003176 were predicted by CircInteractome (https://circinteractome.irp.nia.nih.gov/). The target genes of miR-182-5p were predicted by PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html), miRmap (https://mirmap.ezlab.org/), miRanda (http://bioinfo.au.tsinghua.edu.cn/micrornadb/index.php), and TargetScan databases (http://www.targetscan.org/). The relationship between circRNA and RBM5 was also confirmed by using the GEPIA  online software (http://gepia.cancer-pku.cn/).The circRNA expression profile related to NSCLC was investigated in GSE101684 and GSE112214 data sets from GEO database (μg/mL streptomycin) at 37°C in a humidified atmosphere with 5% CO2. The overexpression plasmid plenti-ciR-GFP-T2A-puro for hsa_circ_0003176 and the short hairpin RNA (shRNA) of RBM5 were designed and constructed to pLKO.1-U6 vector. The sequence of full length of hsa_circ_0003176, the shRNA of RBM5, miR-182-5p mimics, miR-182-5p inhibitor, and their corresponding negative control (NC) were synthesized at Sangon Biotech . All the plasmid or miRNAs mimics/inhibitor were transfected to the cells using Lipofectamine 8000 reagent  according to the protocols provided by the manufacturer. The sequence of all shRNAs, miR-182-5p mimics, miR-182-5p inhibitor, and their negative control is as follows: sh-RBM5#1: 5′-CCGGTCCCAGACCTAAGTTTGAAGATCTCGAGATCTTCAAACTTAGGTCTGGGTTTTTGAATT-3′, sh-RBM5#2: 5′-CCGGTACCATCACAGAGAGCGATATTCTCGAGAATATCGCTCTCTGTGATGGTTTTTTGAATT-3′, sh-RBM5#3: 5′-CCGGTCGCGTCTTTAGCTGTCAATAACTCGAGTTATTGACAGCTAAAGACGCGTTTTTTGAATT-3′, sh-NC: 5′-CCGGTCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTGAATT-3′, NC mimics: sense: 5′-UUGUACUACACAAAAGUACUG-3′ and antisense: 5′-GUACUUUUGUGUAGUACAAUU-3′, hsa-miR-182-5p mimics: sense: 5′-UUUGGCAAUGGUAGAACUCACACU-3′ and antisense: 5′-UGUGAGUUCUACCAUUGCCAAAUU-3′, NC inhibitor: 5′-CAGUACUUUUGUGUAGUACAA-3′, and hsa-miR-182-5p inhibitor: 5′-AGUGUGAGUUCUACCAUUGCCAAA-3′.Human normal bronchial epithelioid cells, 16HBE cells, and H1299, H446, 95-D, and A549 cell lines were purchased at the American Tissue Culture Collection (ATCC). Among H446 cell line is small-cell lung cancer cells, whereas H1299, 95-D, and A549 cell lines are non-small-cell lung cancer cells. These cell lines were cultured in DMEM  supplemented with 10% fetal bovine serum  and double antibiotic  acts as internal control for hsa_circ_0003176 and RBM5, and U6 acts as internal control for miR-182-5p. Relative expression of genes was measured by the 2-△△Ct method. The primer sequences used in RT-qPCR in the present study are as follows: hsa_circ_0003176: forward primer: 5′-GGAGCTGCACAGAATGTCAA-3′ and reverse primer: 5′-CTTTTGCGTCAGGCTCCAG-3′; divergent primer: forward: 5′-GGAGCTGCACAGAATGTCAA-3′ and reverse: 5′-CTTTTGCGTCAGGCTCCAG-3′; convergent primer: forward primer: 5′-ACGTCTTTCCTGAGACTGCG-3′ and reverse primer: 5′-TCATCACTGCCCCGAAATCC-3′; miR-182-5p: RT primer: 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAGTGTGA-3′, forward primer: 5′-CGGCTTTGGCAATGGTAGAAC-3′, and reverse primer: 5′-GTCGTATCCAGTGCGTGTC-3′; RBM5: forward primer: 5′-GACCGATCCGAAGATGGCTAC-3′ and reverse primer: 5′-TTGCTCTCCCTCTCGTCACTG-3′; GAPDH: forward primer: 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse primer: 5′-GCCATCACGCCACAGTTTC-3′; and U6: forward primer: 5′-CTCGCTTCGGCAGCACATATACT-3′ and reverse primer: 5′-ACGCTTCACGAATTTGCGTGTC-3′.The total RNAs from cells and tissues were extracted using TRIzol reagent . For hsa_circ_0003176 and RBM5 expression detection, complementary DNA (cDNA) transcription from RNA was performed using PrimeScript™ RT reagent Kit with gDNA Eraser . For miR-182-5p expression, the RNA was reversed transcribed into cDNA by miRNA 1st Strand cDNA Synthesis Kit . The Real-Time qPCR was conducted on LightCycler®96(Roche) to detect the mRNA abundance of hsa_circ_0003176 and RBM5 with SYBR Green qPCR Mix  and the miR-182-5p with miRNA Universal SYBR qPCR Master Mix  according to the manufacturer's instructions. For the hsa_circ_0003176 identification by RT-qPCR, the total RNA was pretreated with 3 U/μg/ml) for 5 min and then washed with PBS for 3 times. And then, the slides were incubated for 1 h at 37°C incubator with prehybridization solution. Subsequently, the hybridization solution containing the hsa_circ_0003176 probe with concentration at 1 μM was added and hybridize at 42°C overnight. Then, the hybridization solution was washed with 2 × SSC, 1 × SSC, and 0.5 × SSC for 10 min, 5 min, and 10 min at 37°C, respectively. Specific probes of hsa_circ_0003176 (sequence: 5′-GCAAGGTTCTGCTCTTTGCAT-3′) were synthesized at Geneseed Biotech . The immunofluorescence signal was observed by FAM-conjugated anti-digoxin. 4,6-Diamino-2-phenyl indole (DAPI) was used to stain the cell nuclei. Images were taken by an inverted fluorescence microscope  at 488 nm.The cell slides were fixed in 4% paraformaldehyde for 20 min and washed 3 times in phosphate buffered solution  on a destaining shaker. The cells were digested by added proteinase K . And then, the expression levels of hsa_circ_0003176 were detected by RT-qPCR. GAPDH acted as the cytoplasm control, and U6 acted as the nuclear control.μg protein was loaded onto sodium dodecyl sulfate (SDS) gel, and then, the proteins were separated using the 10% SDS-PAGE. Subsequently, transfer protein bands onto the PVDF membranes . Then, the membrane was incubated with 5% skim milk at room temperature for 60 min. Then, the membranes were incubated by primary antibodies against anti-Rabbit RBM5  and anti-Rabbit GAPDH  overnight at 4°C. After washing by PBS buffer for 3 times, the membranes were then incubated with the Goat anti-Rabbit IgG  at room temperature for 70 min. Finally, the protein bands were visualized by Gel imaging system  under ECL regent. The protein band was quantified using the Image J software .The total protein was isolated by the RIPA lysis buffer (Solarbio) according to the instruction procedures. The protein was quantified by bicinchoninic acid (BCA) detection kit . And then, 40 3 cells/well H1299 cells and A549 cells were plated in 96-well plates and subsequently grow in DMEM with 10% FBS for 24 h. 10 μl Cell Count Kit-8 (CCK-8) solution was added to the 96-well plates after transfection, and cells underwent 24 h, 48 h, and 72 h incubation at 37°C in an incubator with 5% CO2. The absorbance  was detected by a spectrophotometer  at 450 nm.Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) Kit  according to the instruction manual. 2 × 10Cell proliferation was also detected by the colony formation assay according to the instruction manual. Briefly, the treated NCSLC cells were seeded onto 6-well plates for 14 days, and the colonies were photographed and counted under the light microscope .μm, Corning) containing the upper and lower chamber which separated by a polycarbonate membrane were seeded in the 24-well culture plate. 3 × 105 cells/mL H1299 cells or A549 cells were prepared by serum-free medium. And then, the upper chamber containing 0.5% bovine serum albumin (BSA) was appended with 200 μL/well. Subsequently, the lower chamber was appended with culture medium containing 10% FBS and cultured for 48 h. Subsequently, the cells in the lower chamber were fixed, dyed, and decolorized. Finally, the images were photographed under the light microscope  and then counted by the Image J software .Transwell chambers  at 0 h and 24 h. Mobility/% = (0 h scratch width − 24 h scratch width)/0 h scratch width.The 3′UTR of RBM5 and hsa_circ_0003176 containing wild type and mutant sequences of binding sites was cloned into pmiGLO vector  to generate pmiGLO-hsa_circ_0003176 vector and pmiGLO-RBM5 vector. And then, the pmiGLO-hsa_circ_0003176 vector or pmiGLO-RBM5 vector was cotransfected to NSCLC cells with miR-182-5p mimics or NC mimics, respectively. The luciferase was detected by Dual-Luciferase Assay System  at 48 h after the transfection following the instruction of manufacturer.The biotin-labeled probes for hsa_circ_0003176 were designed and synthesized by Sangon Biotech . The binding relationship between hsa_circ_0003176 and miR-182-5p was performed by the RNA pull-down assay. Briefly, the cells with were fixed, lysed, and centrifuged, and then, the supernatants were collected. Subsequently, the supernatants were incubated with the biotin-labeled probes for hsa_circ_0003176 overnight at room temperature. After that, the formaldehyde crosslinking was reversed by the lysis buffer and proteinase K. RNA was extracted by TRIzol reagent . The supernatants were used as input, and the expression of hsa_circ_0003176 and miR-182-5p was detected by RT-qPCR.n = 6 mice/group) and lung metastasis experiment (n = 5 mice/group) were carried out in nude mice. First, we constructed A549 cells with stable overexpressing hsa_circ_0003176. Briefly, the A549 cells were pretransfected with overexpressing hsa_circ_0003176 lentivirus, and then, the overexpressing hsa_circ_0003176 A549 cells was filtered with puromycin  to get stable A549 cells. The A549 cells transfected with negative control lentivirus acted as negative control. The transfection efficiency was determined by RT-qPCR. For subcutaneous xenograft experiments, a total of 2 × 106 A549 stable cells with or without overexpressing hsa_circ_0003176 were subcutaneously injected into the right armpit of male nude mice. The tumor volume and the weight of the nude mice were measured on days 3, 7, 14, 21, and 28 after subcutaneous xenograft, and tumor volume was calculated according to the formula volume = length × width2 × 0.5. At the end of feeding, mice were sacrificed with the tumor removed for weighing and histological analysis. For lung metastasis experiment, the nude mice were injected tail vein with a total of 2 × 106 A549 stable cells with or without overexpressing hsa_circ_0003176. At the end of feeding (4 weeks), mice were sacrificed with the lung tissues removed for histological analysis. All the animal experiments were approved by the Ethics Committee of the University of Hong Kong-Shenzhen Hospital .Male BALB/c nude mice  were purchased at Zhuhai Baishitong Biotechnology Co., Ltd (License No.: SCXK (Guangdong) 2020-0051, Animal Certificate No. 44822700003250). After one week of adaptive feeding, subcutaneous xenograft experiments (μm sections. For hematoxylin-eosin (H&E) staining, the sections were stained with hematoxylin and differentiation with hydrochloric acid alcohol. And then, ammonia water was used to immerse the sections for 10-30 s to return to the blue. And then, the sections were stained with eosin solution  and dehydrated with alcohol, xylene permeabilization and neutral gum sealing. After that, observation was performed under the light microscope .The collected tissues were routinely dehydrated, embedded, and cut into 3 For Ki-67 immunohistochemistry, primary antibody anti-Ki67  was used to incubate the sections. After that, the sections were incubated with secondary antibody anti-rabbit horseradish peroxidase-conjugated antibody for 45 min. And then, the slides were stained using 3,3′diaminobenzidine (DAB) solution . Images were taken and analyzed with the light microscope  and Image J software , respectively. The expression level of Ki-67 was calculated as average optical density (AOD) = integrated option density (IOD)/area.t-tests and one-way ANOVA assay. A value of p < 0.05 indicated a statistically significant difference.Statistical analyses in the present study were detected using SPSS 22.0 software (IBM) and GraphPad Prism 8. Each study has at least three biological replicates. Data are shown as means ± standard deviation (SD). Pearson correlation coefficient analysis was used to evaluate correlations between genes. Differences between two groups and more than two groups were analyzed by Student's https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101684) and GSE112214 datasets containing three NSCLC lung samples and three matched adjacent normal samples (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) to analyze the hsa_circ_0003176 expression in NSCLC tissues and matched adjacent normal tissues. The result showed that hsa_circ_0003176 was significantly downregulated in NSCLC tissues compared with matched adjacent normal tissues  were transfected with hsa_circ_0003176 overexpression plasmid alone or cotransfected with miR-182-5p mimics. CCK-8 and colony formation assay indicated that overexpressing miR-182-5p could reduce the proliferation inhibition to both A549 and H1299 cells caused by overexpression of hsa_circ_0003176 Figures . Wound-hcircRNAs can regulate the expression of the target genes of miRNAs by acting as a ceRNA to sponge microRNA . We predTo investigate the directly interaction between miR-182-5p and RBM5, dual-luciferase reporter gene detection was performed with wild-type and mutant RBM5–3′ untranslated region (UTR) vectors incorporating miR-182-5p binding sites (RBM5-wild-type vector and RBM5-mutant-type vector) . The resIn order to know whether hsa_circ_0003176 participated in the regulation of NSCLC cell proliferation, migration, and invasion by regulating RBM5 expression, the designed RBM5 shRNA  was used to silence the expression of RBM5 in both A549 and H1299 cells. The results indicated that all of the shRNAs for RBM5 significantly downregulated the expression of RBM5, and both shRNA#1 and shRNA#2 showed good interference effect Figure . Thus, oLung cancer is one of the common malignant tumors, among which NSCLC accounts for about 80% of the incidence of lung cancer . Most ofCircular RNAs (circRNAs) are a class of endogenously conserved noncoding RNAs that have been shown to be involved in transcriptional and posttranscriptional gene regulation, thereby regulating the occurrence and development of various diseases, including NSCLC , 25, 26.It has been reported in the literature that circRNAs can act as miRNA sponges to regulate the expression levels of miRNAs and downstream target genes , 31. In In addition, it has been demonstrated that RBM5 acts as tumor suppressor in different cancers. Compared with normal tissues, RBM5 was significantly lower in medulloblastoma and RBM5 overexpression repressed cell proliferation and migration in vitro . Zilun eIn this study, our results demonstrated that hsa_circ_0003176 inhibited the growth and metastasis of NSCLC in vitro and in vivo and elucidated that hsa_circ_0003176 regulated the malignant biological functions of NSCLC cells by targeting the expression of RBM5 through sponge adsorption of miR-182-5p in vitro, but hsa_circ_0003176 inhibiting NSCLC progression by regulating miR-182-5p/RBM5 axis has not been verified in animals. In the next step, we will further validate this result in animals, expand clinical samples to verify the clinical significance of hsa_circ_0003176/miR-182-5p/RBM5 signaling axis, and explore the underlying molecular mechanism of hsa_circ_0003176 dysfunction in NSCLC.Our results indicated that hsa_circ_0003176 suppressed the progression of NSCLC via regulating RBM5 expression by sponging miR-182-5p. These findings provided potential therapeutic targets to develop new strategy for NSCLC treatment.