We show for the first time thaturanyl ions bind to Aβ peptides with affinities in the micromolarrange, induce structural changes in Aβ monomers and oligomers,and inhibit Aβ fibrillization. This suggests a possible linkbetween AD and U exposure, which could be further explored by cell,animal, and epidemiological studies. General toxic mechanisms of uranylions could be modulation of protein folding, misfolding, and aggregation.Uranium (U) is naturally present in ambient air, water,and soil,and depleted uranium (DU) is released into the environment via industrialand military activities. While the radiological damage from U is ratherwell understood, less is known about the chemical damage mechanisms,which dominate in DU. Heavy metal exposure is associated with numeroushealth conditions, including Alzheimer’s disease (AD), themost prevalent age-related cause of dementia. The pathological hallmarkof AD is the deposition of amyloid plaques, consisting mainly of amyloid-β(Aβ) peptides aggregated into amyloid fibrils in the brain.However, the toxic species in AD are likely oligomeric Aβ aggregates.Exposure to heavy metals such as Cd, Hg, Mn, and Pb is known to increaseAβ production, and these metals bind to Aβ peptides andmodulate their aggregation. The possible effects of U in AD pathologyhave been sparsely studied. Here, we use biophysical techniques tostudy in vitro interactions between Aβ peptides and uranyl ions,UO Of particularinterest is the effect of uranyl on the Aβ structure and aggregation.Here, we use biophysical spectroscopic and imaging techniquestoinvestigate the binding interactions between uranyl ions and the threeAβ peptide variants Aβ1.175 Regulatory agencies have limitedthe highest allowed concentration of U in drinking water to 30 μg/L,76 replacing a previous WHO limit of 15 μg/L.Absorption of U is low regardless of exposure route and highly dependenton its solubility.39 Inhaled U-dust particlesof low solubility can be retained in tissues for many years.77 Occupational exposure to U has historicallyinvolved workers in the production of phosphate fertilizers, workersproducing glazed pottery, and miners handling uranium oxide, so-called“yellowcake”.78 Sleep disturbancesand possibly encephalitis have been linked to U exposure in formerU mining districts in Kazakhstan.79Uranium is present inambient air, water, and soil. The highest human exposures result fromdrinking well water in geological regions rich in U.82 Uranium also accumulates in bone.85 Uranium crosses the blood–brainbarrier to accumulate in the CNS.50 Here,inhalation and ingestion of U yields heterogeneous but specific accumulations,most prominent in the hippocampus region,86 responsible for memory recall. Rats surgically implanted with Upellets for 6 months have shown the presence of U in the cortex, midbrain,and cerebellum.87 A study on mice showedtoxic effects of DU in the mouse fetus,88 indicating that U can also cross the placental barrier.The main target for U toxicity is the kidney where atrophy and necrosisof glomerular walls have been noted.73 stomachcancer,89 liver or bladder cancer,90 or, as mentioned above, AD.49 One study, however, found cerebrospinal fluid U concentrations,albeit at low overall concentrations, to be significantly elevatedin 17 patients with amyotrophic lateral sclerosis (ALS) when comparedto 10 controls.91 Studies in rodents haverelated U exposure to distorted social behavior92 and weakened sensorimotor behavior.93 Laboratory experiments using organisms such as rats and Caenorhabditis elegans have concluded with a lowacute neurotoxic potential of U following exposure and a protectivepotential from the small metal-regulating protein metallothionein.94 Protective effects have also been observed for the ghrelin hormone95 and for antioxidant agents, including glutathione.97Thereare currently no known correlations between uranium exposureand diseases such as leukemia,22+. This ion is common in uranium-containing minerals, butit is also water-soluble and is present in the ocean at a concentrationof 13.7 nM.98 The uranyl ion is paramagnetic,100 and in aqueous solution, it acts as a weak acid with a pKa of around 4.2.101 It behaves as a hard acceptor and prefers to form complexes withfluoride and oxygen donor atoms, preferably in planar geometry involvingfour, five, or even six binding ligands.102 The capacity to accommodate five or six equatorial ligands in pentagonalor hexagonal bipyramidal coordination separates the uranyl ion frommost other metal ions.98 Thus, these uncommonbinding geometries have been employed in attempts to design uranyl-specificbinding proteins, e.g., to extract uranium from seawater.98 As the most common form of uranium, most experimentalstudies of uranium toxicity have actually been conducted with uranylions rather than with metallic uranium, which is extremely rare innature.The most predominant, most stable, and most relevant formof uraniumin aerobic environments is the uranyl oxycation, UO104 This is consistentwith the ApoEε4 allele of the ApoE gene being a risk factorfor mercury toxicity106 and with ApoE-deficient miceshowing increased iron accumulation in tissue over time.107 It therefore appears likely that the ApoE proteinis involved in regulating metal homeostasis. The ApoEε4 alleleis further linked to an increased probability of developing AD.112 Why the ApoE gene is a risk factor for both heavy metal toxicityand AD is currently unclear, and various explanations have been proposed.114 The previously suggested hypothesisthat ApoE might bind and transport metal ions via Cys residues,106 which are present in ApoE2 and ApoE3 but not in ApoE4, has recentlybeen called into question.115Interestingly, U intoxication and AD appear to havea common riskfactor in the gene coding for apolipoprotein E (ApoE). U exposurein mice was found to induce cognitive impairment in ApoE-deficient(ApoE–/−) males, together with some changes in cholesterollevels and metabolism.22.1116 was monitored when samples of 20 μM Aβ40 peptides in 20 mM MES buffer, pH 7.3, were incubated withshaking for 15 h at 37 °C, together with different concentrations of uranyl acetate  images were recorded for 20 μM Aβnd 20 μM 2. The ur-fibrils 2A. This nd shape 2B. In thnd shape 2C. At thnd shape 2D. Instearticles 2D. These2.340 peptides. 1H, 15N-HSQC spectra for theamide cross-peak region for 92 μM 15N-labeled Aβ40 peptides, at either pH 7.3 or pH 5.1, recorded before andafter the addition of uranyl acetate.High-resolution nuclear magnetic resonance(NMR) experiments were conducted to investigate if there were residue-specificmolecular interactions between uranyl ions and monomeric Aβ40 ratio, respectively)induces a concentration-dependent loss of amide cross-peak intensitychemical exchange between free Aβ40 peptides andthe Aβ40 uranyl complex, similar to the effects observedwhen Cu(II), Ni(II), and Zn(II) ions bind to Aβ peptides.120 In addition, the uranyl ions likely promote the formation of Aβaggregates that either precipitate out of the solution or are toolarge and heterogeneous to produce distinct NMR signals.120 As no NMR signals are observed for the Aβ40 aggregates,nothing can be concluded about the binding configurations for uranylions in complex with such aggregates.At pH 7.3, addition of first 46 μM and then92 μM uranylions (1:2 and 1:1 uranyl/Aβntensity3A. The i40 ratio) at pH 5.1 decreases the cross-peak intensity approximatelyas much as the addition of 92 μM uranyl acetate (1:1 ratio)at pH 7.3  shown in Uranyl ions were found to quench the intrinsicfluorescence of the Tyr10 residue in the Aβ peptide, similarto, e.g., Cu(II) ions.40 at pH 7.3, the titrationdata clearlydeviate from the binding model, both for the measurements in bufferonly  mutantat pH 7.3 follow the model rather well  mutant at pH 5.1 (3.0 μM),while the weakest binding is observed for Aβ40 atpH 7.3. It therefore appears that neutral  Hisresidues might interfere with uranyl binding under the experimentalconditions used.For both peptide versions, the uranyl ions display stronger bindingat low pH. Even though reliable binding constants could not be obtainedfor Aβt pH 5.1 4A,B. Addt pH 5.1 2. This e2.540 peptides, both in aqueous buffer and in thepresence of SDS micelles that mimic a membrane environment. The CDspectra for Aβ40 monomers in aqueous buffer displayspectra with minima around 196–198 nm  spectroscopy was used toinvestigate the possible effects of uranyl ions on the secondary structureof Aβ6–198 nm 5B,E, whiy 214 nm 5E. The dy 214 nm 5F shows y 214 nm 5B. The dy 214 nm 5C.40 peptides display minima around 208 and 222nm  crosses the CD spectrum for Aβ40 peptidesin SDS micelles , when no uranylions are present  ions.118 Two samples of Aβ40 peptides were studied. The control sample contained 100μM EDTA to remove possible contaminating metal ions that couldpromote dityrosine formation. The other sample contained 100 μMuranyl acetate. For both samples, the fluorescence spectra are virtuallyidentical before and after 24 h of incubation  ionscubation 6. Specif2.740 peptidesdo not form stable oligomers, Aβ42 peptides togetherwith SDS detergent were used to create oligomers that remained stablefor over a week. The Aβ42 oligomers were createdwith two different concentrations of stabilizing SDS molecules, 80–100μM of Aβ42 peptides and 0–1000 μMof uranyl acetate, as described in Blue native polyacrylamide gel electrophoresis(BN-PAGE) analysis and Fourier-transform infrared (FTIR) analysis(below) were used to investigate possible effects of uranyl ions onAβ oligomer formation. Because Aβ0.05%SDS, most likelycontain twelve Aβ42 peptides and display a globularmorphology, which is why they are sometimes called globulomers.131 In the presence of different concentrationsof uranyl acetate, no well-defined uniform oligomers were observed.Instead, the bands on the BN-PAGE gel appear smeared, indicating aheterogeneous size distribution of the formed oligomers  without uranyl acetate, larger oligomers witha molecular weight (MW) around 55–60 kD are formed 7, lane 342 oligomers witha MW around 16–20 kDa are formed  yields oligomer bandsthat are weaker at the intermediate uranyl concentrations and weakestat the highest concentration, indicating gradually lower amounts ofstable AβO0.2%SDS oligomers , small Aβe formed 7, lane 7ligomers 7.2.842 oligomers formed with different SDS and uranylconcentrations were studied with FTIR spectroscopy, where the amideI region (1700–1600 cm–1) is very sensitiveto changes in the protein backbone conformation, including differencesin β-sheet structures.135 When prepared in the absenceof uranyl acetate, both types of Aβ42 oligomers  producetwo major bands in the amide I region: a high-intensity band near1630 cm–1 and a smaller one near 1686 cm–1.132 This split pattern of IR bands inthe amide I region is typical for anti-parallel β-sheet structures.138The secondary structures ofAβ0.05%SDS oligomers in PBS buffer,the β-sheet main band shows a band shift due to uranyl acetate:the main band is observed at 1628.3 cm–1 withouturanyl acetate, but shifts down to 1627.8 cm–1 inthe presence of 1 mM (1000 μM) uranyl acetate   ion effects,139 where at both SDS concentrations, the standarddeviation of the band positions for four Li(I) ion concentrations was 0.12 cm–1 and the bandpositions at zero and 10 mM Li(I) differed by less than 0.1 cm–1.For AβO acetate 8A. In coacetate) 8B. This 42 globulomers produced at the lowerSDS concentration are more sensitive to uranyl-induced effects.These observations agree with the BN-PAGE results,which showedclear small oligomer bands for all uranyl concentrations at the higherSDS concentration, but extended smears instead in the presence ofuranyl acetate for the lower SDS concentration. The results indicateagain that the Aβ339 but the underlying molecularmechanisms and a possible role of Uin neurodegenerative diseases remain unclear.91 For AD, several studies have investigated how Aβ peptidesinteract with various metal ions142 and with small cationic molecules.143 We here interpretthe current results on Aβ–uranyl interactions in thelight of this earlier work.Uranium is a well-knownneurotoxicant,3.140 peptides at physiological pH. Instead offorming proper amyloid fibrils, the Aβ40 peptidesform non-fibrillar amorphous aggregates (clumps) already at a 1:1uranyl/Aβ40 ratio , Ni(II), Pb(II), and Pb(IV).143 As small oligomeric aggregatesof Aβ peptides are considered to be the main toxic species inAD pathology,11 the finding that uranyl ionscan modulate Aβ aggregation might be relevant for understandingAD progression and pathogenesis.The ThT fluorescence curves 1 and TEM40 ratio 2. This e3.240 peptides atom and negative Aβ residues, such as Asp1,Asp7, Asp23, Glu3, Glu11, and Glu22. This is similar to Aβ bindingto Mn(II) and Pb(II) ions,140 but different fromnumerous small cationic species that display residue-specific interactionswith Aβ peptides, such as polyamines28 and Cu(II), Ni(II), Pb(IV), and Zn(II) ions.119The NMR results show that uranyl ions have noobvious residue-specific binding to Aβpeptides3. Becaus40 peptides and 3.0 ± 1 μM to the Aβ40 (NoHis) mutant  andZn(II) ions.119 On the other hand, theuranyl ion is a weak acid that undergoes hydrolysis, and the firstpKa is around 4–5.146 Thus, at pH 5.1, the uranyl ions may be mixed with monomeric anddimeric hydroxide species. At pH 7.3, additional species might bepresent, such as the single-charged trimer [(UO2)3(OH)5]+. It is possible that such heterogeneity,which is likely more pronounced at neutral pH, could help explainthe somewhat unexpected uranyl titration results.The results of the NMR measurementsindicate that the Aβ binding of uranyl ions is stronger at acidicpH 3, which) mutant 2. At neu) mutant 2 and als) mutant 4. This i40 at pH 7.3, and the strongest binding is observed for theAβ40 (NoHis) mutant at pH 5.1 (40 (NoHis)mutant (40 peptide and to the Aβ40 (NoHis) mutant. In the latter case, the effect is clearlynot caused by His protonation as these residues are missing in themutant peptide. Thus, the His residues are not the only factor responsiblefor the unusual properties of uranyl-Aβ binding. As discussedabove, formation of uranyl hydroxide species is likely another confoundingfactor. The molecular details of uranyl-Aβ binding should thereforebe further explored in future studies.The Tyr10fluorescence measurements 4 show tht pH 5.1 2. This tt pH 5.1 2. This tt pH 5.1 2, when t)mutant 4.In add147 and as membrane disruption might be one ofthe toxic mechanisms of Aβ aggregates,148 it is important to clarify how Aβ interacts with uranyl ionsalso in a membrane environment. Here, we used SDS micelles as a simplemembrane model. Even though such micelles are different from the phospholipidbilayers present in cell membranes, they do share some propertieswith real membranes, while being much more suitable for various spectroscopymeasurements, including NMR.126 Our fluorescence spectroscopymeasurements showed that the uranyl binding affinities decrease slightlywhen SDS micelles are present in the sample for all Aβ variantsand conditions  bind uranylions mainly via their N-terminal part in a membrane environment. Wemay speculate that when multiple Aβ peptides are present inthe membrane, a single uranyl ion might bind to the N-termini fromtwo or more Aβ peptides, thereby bringing the peptides togetherand promoting aggregation.Because toxic Aβaggregates appear to form in membrane environments,nditions 2. Thise3.340 peptides, fromrandom coil to β-sheet structure  and Ni(II) bind Aβ peptidesmainly via the His6, His13, and His14 residues, it is likely thatthese metal ions can promote Aβ aggregation by coordinatingmultiple His residues from more than one Aβ peptide.149 Cu(II) and Ni(II) ions may therefore promote dityrosine formationnot only by creating oxygen radicals but also by connecting two Aβmolecules and positioning their Tyr10 residues close to each other.It is known that the U(VI) ion in uranyl can be reduced to lower valencystates such as U(IV) under appropriate reducing conditions.150 Thus, as uranyl acetate has been found to induce oxidative stressin isolated cells,97 the uranyl ions arelikely redox-active under physiological conditions. We therefore speculatethat the reason why uranyl ions do not promote dityrosine formationin Aβ40 peptides  ions.118 However, only10 μM of divalent uranyl ions  ions displaya stronger binding affinity for Aβ peptides than uranyl ionsat neutral pH the toxicity of Aβ oligomers or the Aβ-induced pathologyin AD patients. Furthermore, no link has been established betweenuranium/uranyl exposure and AD incidence. On the other hand, veryfew people have investigated such possible links. The current resultsshow that uranyl ions affect Aβ peptide aggregation in similarways as Pb and Hg ions, and Pb and Hg exposure might be linked tothe development of AD and other neurodegenerative diseases.422+, bind to Aβ40 peptides via non-specificelectrostatic interactions, withan apparent binding affinity of 16.3 ± 4 μM at pH 5.1.Uranyl binding is weaker and less uniform at neutral pH, possiblybecause of interference from His sidechains and from other uranylspecies such as hydroxides. The uranyl ions inhibit Aβ fibrillizationand oligomer formation in a concentration-dependent manner, with cleareffects already at sub-stoichiometric concentrations, and also inducestructural changes in Aβ monomers and Aβ oligomers. Ageneral toxic mechanism of uranyl ions could be to modulate proteinfolding, misfolding, and aggregation.Uranyl ions, UO55.1N-morpholino)ethanesulfonic acid (MES) hydrate, sodium phosphate, depleted uranylacetate, and dimethyl sulfoxide (DMSO) were all purchased from Sigma-Aldrich(USA).SDS detergent, 2-. Two recombinant Aβ variants were purchasedas lyophilized powder from AlexoTech AB , namely,the Aβ40 peptide and the Aβ40  triple-mutant, which in the following is referredto as the Aβ40 (NoHis) mutant. The Aβ40 peptide was also purchased uniformly single-labeled with 15N isotopes. All Aβ40 peptide variants were storedat −80 °C until use. Before measurements, they were dissolvedin 10 mM NaOH and then sonicated in an ice bath for 5 min to avoidpre-formed aggregates. The samples were then diluted in either sodiumphosphate buffer at pH 7.3 or in MES buffer at pH 7.3 or 5.1. Thepeptide concentrations were initially estimated from the weight ofthe dry powder and then more accurately determined with a NanoDropspectrophotometer.Synthetic lyophilized wt Aβ (1–42) peptides,abbreviated as Aβ5.242 peptides with low concentrations(≤7 mM) of SDS, i.e., below the critical micelle concentrationfor SDS, which is 8.2 mM in water at 25 °C,152 leads to the formation of stable and homogeneous Aβ42 oligomers of certain sizes and conformations.153 To prepare such oligomers, sizeexclusion chromatography (SEC) was initially used to purify syntheticAβ42 peptides into monomeric form. First, 1 mg oflyophilized Aβ42 powder was dissolved in 250 mL ofDMSO. Next, a Sephadex G-250 HiTrap desalting column  was equilibrated with a 5 mM NaOH solution (pH = 12.3) andwashed with a solution of 10–15 mL of 5 mM NaOD, pD = 12.7.154 The peptide solution in DMSO was applied tothe column, followed by an injection of 1.25 mL of 5 mM NaOD. Thecollection of peptide fractions in 5 mM NaOD on ice was started ata 1 mg/ mL flow rate. Ten fractions of 1 mL volumes were collectedin 1.5 mL Eppendorf tubes. The absorbance for each fraction at 280nm was measured with a NanoDrop instrument , andpeptide concentrations were determined using a molar extinction coefficientof 1280 M–1 cm–1 for the singleTyr in Aβ42.155 The peptidefractions were flash-frozen in liquid nitrogen, covered with argongas on top in 1.5 mL Eppendorf tubes, and stored at −80 °Cuntil used. SDS-stabilized Aβ42 oligomers of twowell-defined sizes (approximately tetramers and dodecamers) were preparedaccording to a previously published protocol,131 but in D2O, at a 4-fold lower peptide concentrationand without the original dilution step.132 The reaction mixtures were incubated together with 0–1000 μM uranyl acetateat 37 °C for 24 h and then flash-frozen in liquid nitrogen andstored at −20 °C for later analysis.Treatment of Aβ5.340 aggregationkinetics, a FLUOstar Omega microplate reader was used. Samples containing 20 μM Aβ40wt,20 mM MES buffer pH 7.3, 50 μM thioflavin T, and different concentrationsof uranyl acetate  wereadded to a 384-well plate, with 35 μL of sample in each well.Thioflavin T is a benzothiazole dye that increases in fluorescenceupon binding to amyloid aggregates116 andis therefore used to monitor the formation of amyloid aggregates.The ThT dye was excited at 440 nm, and ThT fluorescence emission at480 nm was measured every 5 min. Before each measurement, the platewas shaken in orbital mode for 140 s at 200 rpm. The samples wereincubated for a total of 15 h, and the assay was repeated three timeswith four replicates of each condition. To determine kinetic aggregationparameters, the data was fitted to F0 and F∞ are the intercepts of the initial andfinal fluorescence intensity baselines, m0 and m∞ are the slopes of theinitial and final baselines, t1/2 is thetime needed to reach halfway through the elongation phase , and τ is the elongation time constant.116To monitor the effect of uranyl ions on Aβ5.440 peptides that had aggregated under the same conditions as in theThT fluorescence studies (above). Thus, 20 μM of Aβ40 in 20 mM MES buffer, pH 7.3, was incubated for 20 h on athermo shaker at 37 °C and 300 rpm, together with 0, 0.2, 2,and 20 μM of uranyl acetate. Then, samples of 5 μL wereput on copper grids of 200 μm mesh size, which were coveredwith a Pioloform film upon which a carbon layer had been depositedand then glow-discharged with a Leica EM ACE600 carbon coater . The Aβ40 samples were absorbedonto the grids for 5 min, rinsed with Milli-Q water two times, andthen stained for 2 min with a 2% aqueous solution of uranyl acetate.Next, the excess stain was removed with filter paper, and the sampleswere left to air-dry. A digital Orius SC1000 camera was used to recordTEM images in a FEI Tecnai G2 Spirit electron microscope  operating at 120 kV accelerating voltage.Negative staining TEM images were recorded for Aβ5.540 peptide in 20 mM phosphate buffer, at either pH 7.3 or pH 5.1, wereplaced in a quartz cuvette with a 2 mm pathlength. CD spectra wererecorded at 20 °C between 192 and 250 nm using steps of 0.5 nmand a sampling time of 5 s per data point. Then, small volumes ofuranyl acetate were titrated to the samples in steps of 2, 6, 16,56, and finally 256 μM. The total increase of volume upon theaddition of the uranyl acetate was less than 3%. All data was processedwith the Chirascan Pro-Data v.4.4.1 software , including smoothing with a ten-point smoothing filter.50 mM SDS detergent was added to some of the samples as SDS micellesconstitute a simple model for bio-membranes.126 Aβ peptides are known to insert their central and C-terminalsegments as α-helices into SDS micelles, while the N-terminalAβ segment remains unstructured outside the micelle surface.125 Because the critical micelle concentration for SDS is 8.2 mM inwater at 25 °C,152 micelles clearlyformed under the experimental conditions. With approximately 62–65SDS molecules per micelle,156 50 mM SDSyields a micelle concentration slightly below 1 mM, i.e., much higherthan the concentration of Aβ peptides. This means that eachmicelle will generally contain no more than one Aβ peptide,which effectively prevents Aβ aggregation and ensures that uranylinteractions are with monomeric Aβ peptides. The high SDS concentrationused to obtain this condition, i.e., 50 mM, does not pose a problemin the current experiments. Even though 50 mM SDS would efficientlydenature most folded proteins, such denaturing effects are not relevantfor small intrinsically disordered peptides such as Aβ (in monomericform).CD measurements were carried outon a Chirascan CD spectrometer from Applied Photophysics Ltd. (U.K).Samples containing 600 μL of 10 μM Aβ5.640 peptide in 20 mM MESbuffer, pH 7.3. One sample contained 100 μM uranyl acetate toinvestigate the possible effects of uranyl ions on dityrosine formation.The control sample contained 100 μM of the chelator EDTA toremove any free metal ions. All measurements were conducted in triplicateusing a quartz cuvette with a 4 mm path length and containing a 0.7mL liquid sample. Spectra were recorded after 0 and 24 h of incubation,during which the samples were kept at room temperature without agitationor other treatment.Fluorescence emission spectra between 330 and 480 nm (excitationat 315 nm) were recorded at room temperature with a Jobin Yvon HoribaFluorolog 3 fluorescence spectrometer  for twosamples of 10 μM Aβ5.715N-labeled Aβ40 peptides in20 mM MES buffer (90/10H2O/D2O) at either pH7.3 or pH 5.1 at 5 °C. Two-dimensional 1H, 15N-HSQC (heteronuclear single quantum coherence) NMR spectra wererecorded during the titrations using settings with 128 t1 increments,24 scans, and a 1 s recycle delay. The spectra were then processedand evaluated in the Topspin software (v. 3.2) using already publishedassignments for HSQC cross-peaks of Aβ40 in bufferat neutral pH159 or at acidic pH.119NMR spectroscopy experiments were conducted on a Bruker Avancespectrometer operating at 500 MHz and being equipped with a cryoprobefor increased sensitivity. Uranyl acetate was titrated to 92 μMmonomeric 5.840 peptides were evaluated via the quenchingeffect of uranyl on the intrinsic fluorescence of Tyr10, the onlynatural fluorophore in the wt Aβ peptide. Fluorescence measurementswere conducted on a Jobin Yvon Horiba Fluorolog 3 fluorescence spectrophotometer using a quartz cuvette with a 4 mm path length.The fluorescence emission intensity of samples containing 20 μMAβ peptides in 20 mM MES buffer, at either pH 7.3 or pH 5.5and without or with 50 mM SDS detergent present, was measured at 305nm (excitation wavelength 276 nm) at 20 °C. Aliquots of uranylacetate  were titrated tothe samples, and the Tyr10 fluorescence intensity was plotted againstthe concentration of UO22+ ions. Apparent dissociationconstants (KDApp) were determinedby fitting the plots to eq I0 is theinitial fluorescence intensity with no added UO22+ ions, I∞ is the steady-stateintensity at the end of the titration, [Ab] is the protein concentration,and [U] is the concentration of added UO22+ ions.160The binding affinities between uranylions and Aβ5.942 peptides132 prepared . The gels were run at 4 °C using the electrophoresissystem according to the Invitrogen instructions  and then stained with the Pierce Silver Staining Kit accordingto the manufacturer’s instructions . BN-PAGE was chosen for analysis instead of SDS–PAGE toavoid disruption of the SDS-stabilized and non-cross-linked Aβ42 oligomers by the high (>1%) SDS concentrations used inSDS–PAGEsample buffers.161Homogeneous solutions of oligomers of 80–100 μM Aβ5.1042 oligomers  was positioned in the reference holder. Thesample shuttle was used to measure the sample and reference spectrawithout opening the chamber. The samples were allowed to sit for atleast 15 min after closing the chamber lid to avoid interference fromwater vapor. FTIR spectra were recorded at room temperature in the1900–800 cm–1 range, with 300 scans for bothbackground and sample spectra, using a 6 mm aperture and at a resolutionof 2 cm–1. The light intensities above 2200 cm–1 and below 1500 cm–1 were blockedwith a germanium filter and a cellulose membrane, respectively.162 The spectra were analyzed and plotted withthe OPUS 5.5 software, and second derivatives were calculated witha 17 cm–1 smoothing range.FTIR spectraof the SDS-stabilized Aβ


Methods: The mebeverine hydrochloride (MBHCl) content of commercial formulations was determined in this study using a unique factionalized CeO2/NiO-nanocomposite-coated membrane sensor. Results: Mebeverine-phosphotungstate (MB-PT) was prepared by adding phosphotungstic acid to mebeverine hydrochloride and mixing with a polymeric matrix  and plasticizing agent o-nitrophenyl octyl ether. The new suggested sensor showed an excellent linear detection range of the selected analyte at 1.0 × 10−8–1.0 × 10−2 mol L−1 with regression equation EmV = (−29.429 ± 0.2) log [MB] + 347.86. However, the unfunctionalized sensor MB–PT displayed less linearity at 1.0 × 10−5–1.0 × 10−2 mol L−1 drug solution with regression equation EmV = (−26.603 ± 0.5) log [MB] + 256.81. By considering a number of factors, the applicability and validity of the suggested potentiometric system were improved following the rules of analytical methodological requirements. Conclusion: The created potentiometric technique worked well for determining MB in bulk substance and in medical commercial samples.Background: The remarkable properties of nickel oxide (NiO) and cerium oxide (CeO Nanomaterials have recently been discovered as potential essential components in sensors, material development, medicinal applications, and cancer treatments targeting systems. In order to develop numerous sensitive sensors in our contemporary lives, scientists are working to broaden their field of study . Numerou2NPs) have attracted a great deal of interest because of their outstanding catalytic activity. This has resulted from the rapid and efficient mutation of the oxidation state between Ce4+ and Ce3+, with potential applications in bioanalysis . H. H2NPs, t 350 nm e. It is unctions . The dir2NPs , 31.71° (2 0 0), 47.70° (2 2 0), 56.92° (3 1 1), 76.90° (331), and 79.2° (420), relating to the face-centered cubic CeO2 crystalline structure. These values were in agreement with those in the JCPDS card no.34-0394 for CeO2  on a Cu-k XRD- diffractometer. The XRD diffraction study of CeOfor CeO2 a 42]. T. T2NPs dfor CeO2 b showed 2/NiO nanocomposite showed three low intensity peaks, corresponding to 37.26° (1 1 1), 34.50° (2 0 0), and 63.08° (2 2 0) of NiO nanoparticles. However, various peaks were observed at 2θ values of 28.72° (1 1 1), 31.71° (2 0 0), 47.71° (2 2 0), 56.91° (3 1 1), and 76.90° (4 2 0), corresponding to CeO2NPs, revealing the successful formation of CeO2/NiO nanocomposite  and the MB ions in the analytical sample. The addition of o-NPOE as a plasticizer in the preparation of the coated membranes enhances the flexibility and durability of the membrane. Moreover, the presence of a plasticizer can act as an ionophore. This can be attributed to the donation sites and functional groups present in the plasticizer, which can affect the chelation of the primary analyte ion [2/NiO nanocomposite sensors was measured and is summarized in −5 to 1.0 × 10−2 and 1.0 × 10−8 to 1.0 × 10−2 mol L−1 for the above-described sensors, respectively  separately, with respect to their selectivity towards the detection of MB+ ions. Various foreign materials, including cations , amino acids (histidine and glycine), sugars (lactose and sucrose), and pharmaceutically formulated additives  were tested. The obtained results exhibited excellent selectivity for functionalized MB-PT-CeO2/NiO nanocomposite. The addition of CeO2NPs and NiONPs enhanced the selectivity of the tested drug due to the smaller particle size and the distinct physical and chemical properties of the metal oxide nanoparticles. The free energy of MB+ transfer between the aqueous and active sites in the coated membrane is frequently used to characterize the selectivity of designed MB-PT and MB-PT-CeO2/NiO nanocomposite membrane sensors. No interference was recorded due to the difference in ionic size, mobility, and the permeability of interfering ions compared to MB+  in the potentiometric assay could be estimated when the potential readings of the designed sensors dropped by 17.9 mV. The LOD was estimated to be 2.5 × 10To ensure the accuracy of the developed technique, nine authentic MB sample solutions were analyzed using the proposed sensors. The accuracy was expressed as mean percentage recoveries (Mean ± SD). The findings were 99.65 ± 0.3% and 99.82 ± 0.2%, as shown in Application of intermediate precision (intra-day and inter-day) assays to determine the degree of precision of the developed functionalized potentiometric system was performed. The estimated results were expressed as relative standard deviation percentage (RSD%). The findings were 0.4% and 0.2% for intra- and inter-day assays, respectively, revealing excellent precision (˂2%), as summarized in The robustness of the design system was determined by changing the pH to 8 ± 1 using phosphate; the findings were calculated to be 98.96 ± 0.8 and 99.87 ± 0.4% for the above two mentioned sensors, respectively. Furthermore, the reliability of the current technique was evaluated using another pH meter (Jenway-3510). The obtained data were 98.70 ± 0.6% and 99.74 ± 0.3% for the tested sensors. These results revealed no significant difference between the obtained results and those obtained from the analytical studies.®200 mg/capsule using the MB-PT and MB-PT-CeO2/NiO nanocomposite sensors. The estimated results were found to be 98.72 ± 0.6 and 99.56 ± 0.4 for the two sensors, respectively  and rice (Oryza sativa) husk extracts were used throughout this study to synthesize green metal oxide nanoparticles . All pure grade materials and reagents were purchased from Sigma-Aldrich , including cerium nitrate hexahydrate (99.9%), nickel nitrate hexahydrate (99.9%), phosphotungstic acid hydrate (99.9%), polymeric material , fluidizing materials , as well as other solvents, such as tetrahydrofuran , methanol (99.9%), ethanol (99.9%), and acetone (99.9%). Pure grade mebeverine hydrochloride was supplied from Sigma-Aldrich . The pharmaceutical product Duspatalin® capsules (200 mg/capsule) was purchased from a local pharmacy . Two naturally prepared wheat . The pH optimization was achieved by another pH meter . The characterization of the synthesized nanomaterials was performed using a Perkin-Elmer BX spectrometer , a Shimadzu X-ray-6000 diffractometer , JSM-7610, and a scanning electron microscope , connected with energy-dispersive X-ray spectroscopy .The husks of wheat and rice were collected separately and sieved to remove any tainted materials. Then, six times distilled water was used to wash out any remaining contaminants from the collected husks. The cleaned husks were dried for 24 h at 70 °C in a hot air oven. About 30 g of the powdered dried husks (50–200 µm) was heated in 300 mL of distilled water at 60 °C for two hours under continuous magnetic stirring. Then a Whatman filter paper No. 40 was used for the filtration process. The extracted material was kept in an Erlenmeyer flask at 4 °C to be used later in the production of the metal oxide nanomaterials. Then the material was filtered using a filter (Whatman paper No. 40). The obtained extract of each husk was stored in an Erlenmeyer flask at 4 °C for further use in the synthesis of metal oxide nanomaterials. 2NPs and NiONPs, approximately 50 mL of wheat (Triticum aestivum) and rice (Oryza sativa) husk extracts was heated separately to 60 °C for 30 min; then 50 mL of 1.0 mol L−1 of cerium nitrate hexahydrate and nickel nitrate hexahydrate was added to the heated extracts, respectively. A few drops of 2.0 mol L−1 sodium hydroxide were added dropwise to the above mixtures and continuously heated at 80 °C for 2 h under constant stirring until the formation of CeO2NPs and NiONPs. The formed nanoparticles were washed three times using deionized water to remove excess sodium hydroxide. The obtained nanoparticles were then filtered using Whatman filter paper No.1 and oven dried at 100 °C for 4 h. The synthesis of nanomaterials is illustrated in The phytocomponents of wheat and rice husk extracts, such as cellulose, hemicellulose, lignin, starch, proteins and fatty acids, act as reducing as well as stabilizing agents . To synt−8–1.0 × 10−2 mol L−1 using distilled water was performed from aqueous 1.0 × 10−2 mol L−1 of MB drug solution (0.466 g/100 mL w/v).Serial dilution of MB drugs in the concentration range of 1.0 × 10−2 mol L−1). The resulting electroactive material MB-PT was washed 3 times with distilled water, collected by filtration using Whatman filter paper No.1 and kept aside overnight at ambient temperature for drying.The electroactive material MB-PT was obtained by mixing equal volumes and concentrations of each MB drug solution and the precipitating agent PTA , MB-PT (10 mg), and o-NPOE (0.4 mL) with the organic solvent THF (5 mL). The blended mixture was poured into a rounded 3 cm diameter Petri dish and allowed to dry at ambient temperature until the formation of an oily membrane cocktail. A pure Al wire was polished and washed with acetone 3 times and then dipped in the previously prepared membrane cocktail to fabricate the coated sensor MB-PT. The functionalized mixture was prepared by the same above-mentioned procedure but approximately 10 mg of CeO2/NiO nanocomposite was added to the membrane cocktail under magnetic stirring for 10–15 min to obtain a homogeneous dispersed membrane mixture. After coating the Al wire using the functionalized mixture, the sensors were hung to dry overnight at room MB-PT-CeO2/NiO nanocomposite sensor temperature, then preconditioned by soaking the fabricated sensors in 1.0 × 10−10 mol L−1 MB solution for 12 h. 2/NiO nanocomposite sensor.The membrane cocktail of MB-PT and MB-PT-CeO−8 to 1.0 × 10−2 mol L−1.The linear concentration range of the suggested new sensors can be graphed by plotting the potential readings (mV) of the sensors vs. -logarithm of the MB concentrations using the designed indicator sensor and the reference (Ag/AgCl) electrode. The tested MB solutions were 50 mL each of the concentration range 1.0 × 10−4 mol L−1 of MB solution (50 mL) was tested using the designed sensors by changing the pH from acidic to alkaline values. The pH of the test solution was acidified using a few drops of 0.1 mol L−1 of hydrochloric acid and then elevated using the same concentration of sodium hydroxide. A combined glass electrode was conjugated with a potentiometric system and the pH values vs. the potential readings (mV) were plotted to estimate the independent pH values for the designed sensor [Various factors that affect the potential readings of sensors should be investigated and optimized, including the pH, selectivity, and response time. The pH of 1.0 × 10d sensor . The selectivity of the applied sensor in analytical detection is the most important factor which may affect the potential response of the sensor. The selectivity coefficient was determined to see how much a foreign substance would influence the sensor response to its main ion. The selectivity coefficients were then determined using the separate solutions method (SSM) in accordance with IUPAC rules .−3 mol L−1 of each MB solution and possible foreign substances separately, such as particular cations, sugars, and polysaccharides, amino acids, and coformulation additives. The tolerable values were calculated using the previously reported equation [−3 mol L−1 MB, E2 is the electrode potential of interfering species, Bz+ is the interfering species, and S is the slope of the calibration graph.Briefly, the selectivity coefficient equation .Log Kpo−8 to 1.0 × 10−2 mol L−1 of the MB solutions.The response time was estimated by recording the potential response of 1.0 × 10® capsules (200 mg MB/capsule) was collected and weighed. A standard solution of 1.0 × 10−2 mol L−1 MB (0.466 g/100 mL w/v) was prepared in distilled water. The desired analytical samples were prepared by diluting the previously prepared standard to obtain 1.0 × 10−9 to 1.0 × 10−2 mol L−1 using distilled water. Each MB concentration was measured separately using the designed potentiometric systems MB-PT and MB-PT-CeO2/NiO nanocomposite; the regression equation was used to estimate the nominal content of MB.The content of ten Duspatalin2/NiO nanocomposite sensors for the determination of mebeverine hydrochloride in authentic and commercial products. The advanced and unique characteristics of the functionalized metal oxide CeO2/NiO-nanocomposite-coated membrane sensors displayed excellent detection of MB with respect to the percentage recoveries, linear concentration range, precision, and the limit of detection. When comparing the potential readings of modified sensors to those of regular sensors in terms of sensitivity and selectivity, the created sensors performed better than existing traditional sensors. Additionally, the measurement of the chosen medication was carried out with good selectivity and over a broad linear concentration range with a low limit of detection due to the use of coated-membrane modifiers with metal oxide nanoparticles. The metal-oxide-enhanced membrane sensors can, therefore, be used by pharmaceutical companies, hospitals, and research laboratories to monitor pantoprazole sodium on a regular basis.The current study proposed MB-PT and MB-PT-CeO


Nonsyndromic cleft lip and/or without cleft palate (NSCL/P) with or without hypodontia is a common developmental aberration in humans and animals. This study aimed to identify the loss of heterozygosity (LOH) involved in hypodontia and NSCL/P pathogenesis.This is a cross-sectional study that conducted genome-wide copy number analysis using CytoScan 750K array on salivary samples from Malay subjects with NSCL/P with or without hypodontia aged 7–13 years. To confirm the significant results, simple logistic regression was employed to conduct statistical data analysis using SPSS software.FAF1, WNT3A and BMP5, respectively. There was a significant association between the D1S197 (1p33-32.3) markers containing the FAF1 gene among NSCL/P subjects with or without hypodontia compared with the noncleft subjects .The results indicated the most common recurrent copy neutral LOH (cnLOH) observed at 1p33-1p32.3, 1q32.2-1q42.13 and 6p12.1-6p11.1 loci in 8 (13%), 4 (7%), and 3 (5%) of the NSCL/P subjects, respectively. The cnLOHs at 1p33-1p32.3 (D1S197), 1q32.2-1q42.13 (D1S160), and 6p12.1-6p11.1 (D1S1661) were identified observed in NSCL/P and noncleft children using microsatellite analysis markers as a validation analysis. The regions affected by the cnLOHs at 1p33-1p32.3, 1q32.2-1q42.13, and 6p12.1-6p11.1 loci contained selected genes, namely The results supported the finding that the genetic aberration on 1p33-32.3 significantly contributed to the development of NSCL/P with or without hypodontia. These results have an exciting prospect in the promising field of individualized preventive oral health care. Nonsyndromic cleft lip and/or without cleft palate (NSCL/P) with or without hypodontia is one of the most common congenital disabilities worldwide . Cleft lVarious genetic approaches have been employed to identify genes and genomic loci that contribute to the development of CL/P and hypodontia, including animal model expression studies, candidate gene sequencing, and genome-wide association studies. Recently, the role of chromosomal deformities in genetic defects and diseases has been investigated . Copy nuThis comparative cross-sectional study included 81 individuals (61 NSCL/P cases and 20 noncleft cases) aged 7–13 years. Patient samples were obtained from one of the tertiary hospitals in northeastern Malaysia between 2016 and 2018. The inclusion criteria were children with NSCL/P aged 7–13 years while, those with a cleft palate and patients with other syndromes such as ectodermal dysplasia or Axenfeld–Rieger syndrome were excluded. The comparison group consisted of noncleft children with their age ranging between 7 and 13 years. Based on examination and history taking, healthy children with no orofacial cleft deformities and no family history of NSCL/P were also included. However, those who had undergone orthodontic treatment were excluded from the study. This study has a sample population and design similar to those of the previous study . The samSaliva samples were collected from each patient and stored in sterile 50 mL conical tubes. Genomic DNA was extracted from the samples according to the GeneAll Blood SV Mini Kit manual , which included lysis, binding, washing and elution.oC and 70 °C for 3 h and 20 min, respectively. Amplicon was generated by performing polymerase chain reaction (PCR) on the adaptor-ligated DNA using reagents provided by Affymetrix with the following settings: 94 °C for 3 min, then 30 cycles of 94 °C for 30 s, 60 °C for 45 s, and 65 °C for 1 min, followed by extension at 68 °C for 7 min. The PCR products were then purified and fragmented with DNase 1 for 35 and 15 min at 37 °C and 95 °C, respectively. Subsequently, the fragmented DNA was labeled with biotinylated for 4 h at 37 °C. Fragments of DNA (200 µg) were hybridized with pre-equilibrated Affymetrix Chip CytoScan 750K at 50 °C for 18 h. The arrays were then washed and stained using GeneChip Fluidics Station 450 and Affymetrix GeneChip Command Console Software, followed by data analysis using Affymetrix Chromosome Analysis Suite (ChAS) software.Genome-Wide Human CytoScan 750K Array  was used to analyze genomic alterations according to the manufacturer’s protocol. Genomic DNA (250 ng) was digested with NspI for 2 h at 37 °C. The digested DNA was ligated with adaptors at 16CytoScan750K array technology was used to define allelic imbalances in LOH. LOHs were recognized accordingly using the ChAS software based on the graph of the allelic difference produced. Areas of LOHs are represented by the absence of plots at the value of 0. Copy number status was checked to categorize the allelic imbalances into conventional LOH (CN = 1), copy-neutral LOH , or LOH with complimentary copy number gains (CN ≥ 3).Microsatellite DNA loci are valuable markers for LOH detection. The significance of LOH results obtained from the CytoScan 750K array were undergo validated analysis using microsatellite marker analysis. LOH is described as the loss of one allele at a heterozygous location, causing the cell to have only one version of a gene. In this study, the results of the CytoScan 750K array indicated that genomic areas such as FAF1 (1p33-32.3), WNT3A (1q32.2-42.13), and BMP5 (6p12.1-11.1) were analyzed for cnLOH. Three informative microsatellite markers  located on chromosomes 1p33-32.4, 1q32.2-42.13, and 6p12.1-11.1 were used in this study. Primers were designed and one primer from each pair was end labeled with a fluorescent dye. Table Each PCR product (1 µL) was mixed with 0.25 µL of GeneScan 400HD ROX Size Standard . Each sample was denatured at 95 °C for 5 min and then chilled on ice for 10 min. Capillary electrophoresis analysis was conducted on an ABI 3100 Genetic Analyzer and data were analyzed using GeneScan Analysis 3.7 software. The results could be classified as loss of one allele or LOH, appearance of both alleles (retention of heterozygosity), or involvement of only one allele (noninformative [NI]).Statistical analysis was conducted using SPSS version 26.0. LOH frequency in selected markers was compared between NSCL/P and noncleft subjects with or without hypodontia using simple logistic regression analysis to determine significant chromosome alterations.This study included 81 subjects (61 NSCL/P and 20 noncleft cases) aged between 7 and 13 years. Their salivary samples were subjected to genetic aberration assay using the CytoScan 750K array. The mean (standard deviation) ages of the NSCL/P and noncleft subjects were 10.7 ± 1.83 and 9.9 ± 2.1, respectively. Besides CNVs, genome-wide array technology was also used to describe allelic imbalances in LOH. LOHs were recognized accordingly using the ChAS software based on the graph of the allelic difference produced  exhibited allelic imbalances with copy number losses, and 309 (70%) were LOHs without change in the cnLOH. Of all the allelic imbalances, the most common recurrent cnLOH was observed at 1p33-1p32.3, 1q32.2-1q42.13, and 6p12.1-6p11.1 loci in 8 (13%), 4 (7%), and 3 (5%) NSCL/P cases, respectively. The percentages of cnLOH among NSCL/P subjects with or without hypodontia were 18% (17p11.2), 18% (20q11.21-20q11.23), and 12% (22q13.1-22q13.2)  sample, and each marker was interpreted based on the presence or absence of the correct allele (peak size). The presentation of microsatellite markers with two distinct alleles in the normal control or the NSCL/P and noncleft subjects with or without hypodontia is considered an informative result, whereas the presence of only one allele in both patients and normal control indicates an NI result. LOH is defined as the complete disappearance of one allelic peak from the patient’s DNA or the reduction of the signal intensity  by < 50% in the patient’s DNA compared with the paired normal DNA pattern.Figure p-value of 0.023 , occurs when a maternal  or paternal  chromosome or chromosomal region is duplicated and the other allele is lost . In thisFAF1 encodes the FAF1 protein that binds to the FAS antigen and initiates apoptosis [FAF1 has been demonstrated to play a critical role in normal neuronal cell survival and development, whereas FAF1 downregulation has been linked to numerous aspects of carcinogenesis [Microsatellites, also known as short tandem repeats, is a genetic variant unique to specific chromosomes and sites that can be used to validate LOH and cnLOH. Due to their rapid mutation, large number of alleles, and frequency in genomic DNA, microsatellites are particularly important molecular markers in population genetics, genome mapping, taxonomic study, linkage analysis, genetic fingerprinting, and diversification . The cnLpoptosis . FAF1 haogenesis .FAF1 in signaling pathways critical for normal development and cancer. The loss of peaks at 141 bp indicated the loss of one allele in the sample with LOH on 1p33-1p32.3 and was evaluated as cnLOH. Most subjects required two peaks representing 137 and 141 base pairs. The FAF1 gene was selected from the LOH area as it regulates CNC. CNC is a migrating cell population that forms most craniofacial bones [FAF1 has been found to play an important role in normal neuronal cell survival and proliferation, whereas FAF1 downregulation may play a role in cancer through a variety of mechanisms [FAF1 blocks Wnt signaling by increasing catenin breakdown [FAF1 interacts with the transforming growth factor (TGF)-β type II receptor, inhibiting TGF signaling and thus suppressing epithelial-to-mesenchymal transition (EMT) [An ongoing research is expected to shed light on the role of al bones . Tooth aal bones . FAF1 hachanisms . FAF1 blreakdown . Activatreakdown , 36. Theon (EMT) .WNT3A also contributed to the formation of NSCL/P and hypodontia. This statement is supported by the findings of functional studies and experimental examination of relevant animal models that validate the impact of the WNT signaling system on tooth development and regulation of adult tooth homeostasis [The D1S160 marker was used to confirm LOH on chromosome 1q32.2-1q42.13, which includes the WNT3A gene in 20 NSCL/P and noncleft individuals with or without hypodontia. The CytoScan 750K assay indicated that chromosome 1q32.2-1q42.13 was selected as one region of interest with LOH related to NSCL/P with hypodontia. This LOH region was observed in five NSCL/P samples. This modification does not affect the copy number, which is known as cnLOH. LOH validation was performed through microsatellite marker (D1S160) analysis to confirm the 1q32.2-1q42.13 regions with high probability of LOH detected by CytoScan 750K array. The loss of peaks at 203 bp indicated the loss of one allele and was evaluated as LOH in the sample with LOH on 1q32.2-1q42.13. The typical subjects must have two peaks representing 203 and 223 base pairs. The WNT3A gene belongs to the Wnt gene family, which plays an important role in prenatal development. The WNTA genes code for proteins involved in the chemical signaling pathways of the body. These pathways control the activity of certain genes and cell interactions throughout embryonic development . In the eostasis .BMP5 has been reported to contribute to palatogenesis and odontogenesis [BMP5 may play a role in the development of certain malignancies. The BMP signaling pathway regulates various developmental processes such as cell proliferation, apoptosis, differentiation, and morphogenesis [BMP5 function through the TGF-β pathways. This is because the TGF-β superfamily includes activin, TGF-β, and BMP cytokines. Chi-squared tests were conducted to determine the significance of the association of the LOH of selected markers with NSCL/P and noncleft with or without hypodontia. The LOH of D1S160 (1q32.2-42.13) and D6S1661 (6p12.1-11.1) was not found to be associated with NSCL/P and noncleft palate with or without cleft palate. However, there was a significant association between D1S197 (1p36.33-32.3) markers and a p-value of 0.023.The 6p12.1-6p11.1 locus represented an attractive genomic region in which cnLOHs occurred. cnLOH at 6p12.1-6p11.1 recurred at frequencies of 3% and 2% in NSCL/P subjects with hypodontia and noncleft subjects with hypodontia, respectively. This finding suggests that genes residing at this genomic locus are involved in the regulation of programmed cell death, migration of epithelial cells to the oral or nasal side of the palate, and EMT and disturbances during the early stages of tooth development. Among the specimens examined in this study, LOHs at 6p12-6p11.1 were found to affect eight genes. Of these, ogenesis . This geogenesis . This suThis study has few limitations that need to be acknowledged for future work. First, the small sample size could limit the statistical power of the analysis. Therefore, future studies are recommended to use a larger number of samples and, whenever possible, include an independent cohort of cases and control from different tertiary centers for the result validation. Additionally, discussions should be made on the mechanism underlying the risk association of genes in the region contributing to LOH based on available literature. Future studies should include appropriate experimental works such as chromatin immunoprecipitation assay to explore the interactions between genes and proteins to uncover the actual mechanism behind the associations. Additionally, for the gene expression study, analysis at the protein level could be considered in the future because genes would eventually be translated into proteins, and posttranslationally modified proteins are the actual molecules of the cells. Future studies should also try establishing a correlation between genomic abnormalities  and gene expression changes in NSCL/P and hypodontia to provide novel insights into the mechanism of palatogenesis and odontogenesis.Herein, a genome-wide analysis was conducted to investigate LOHs in NSCL/P patients and uncover probable NSCL/P with hypodontia-related genes. We found three recurrent LOHs in the chromosome at 1p33-1p32.3, 1p32.2-1q42.13, and 6p12.1-6p11.1 loci in NSCL/P subjects with or without cleft palate, whereas 1p33-1p32.2 and 6p12.1-6p11.1 were commonly detected in noncleft subjects without hypodontia. Our findings add to the pool of potential causal markers for NSCL/P and hypodontia for future genetic investigations. Human mutational analysis and animal model research are warranted to confirm the role of the identified putative causal genes of NSCL/P and hypodontia.


The major isozymes identified by zymograms during the first 12 h were 51- and 140-kDa carboxymethyl cellulases (CMCases) and 23- and 57-kDa xylanases. The band densities of 37-, 53-, and 58-kDa CMCases and 38-, 44-, and 130-kDa xylanases increased from 24 to 36 h. A microbial analysis indicated that the relative abundances of Prevotella, Fibrobacter, and Bacteroidales RF16 bacteria, Neocallimastix and Cyllamyces fungi, and Dasytricha and Polyplastron protozoa were related to fibrolytic isozyme activity. The present results provide novel insights into the relationships between fibrolytic isozymes and rumen microorganisms during lignocellulose biodegradation.Rumen fibrolytic microorganisms have been used to increase the rate of lignocellulosic biomass biodegradation; however, the microbial and isozymatic characteristics of biodegradation remain unclear. Therefore, the present study investigated the relationship between rumen microorganisms and fibrolytic isozymes associated with lignocellulosic biomass hydrolysis. Rice straw, a widely available agricultural byproduct, was ground and used as a substrate. The biodegradation of rice straw powder was performed anaerobically in rumen fluid for 48 h. The results obtained revealed that 31.6 and 23.3% of cellulose and hemicellulose, respectively, were degraded. The total concentration of volatile fatty acids showed a 1.8-fold increase (from 85.4 to 151.6 mM) in 48 h, and 1,230.1 mL L All experiments were approved by and performed in accordance with the regulations of the Institutional Animal Care and Use Committee of Tohoku University . All methods were conducted in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.ad libitum. Collected rumen fluid was filtered through a 1×1 mm mesh to remove coarse solids. The total solid content of filtered rumen fluid was 7.1 g L–1.Rumen fluid was collected 2 h post-feeding using a stomach tube from a lactating Holstein cow. Cows were fed a diet consisting of 64% timothy grass and 36% concentrate and given water powder consisted of 34.7% cellulose, 24.1% hemicellulose, 4.4% lignin, and 36.8% of other constituents.Rice straw was collected from rice fields with permission from the Field Science Center, Graduate School of Agricultural Science, Tohoku University . Straw was dried at 60°C overnight, chopped into lengths of 1–2 cm using scissors, and then ground in a laboratory mill  for approximately 5 min. The powder was sieved through a 1×1 mm mesh. This Four grams of the rice straw powder was mixed with 200 mL of rumen fluid and purged with nitrogen gas to remove oxygen. Buffers  were not used in the present study because the cost of reagents is a feasible barrier to the large-scale operation of rumen fluid bioconversion systems. The biodegradation of rice straw powder was performed in a 250-mL reactor equipped with an aluminum gas bag at 37°C on a rotary shaker at 170 rpm for 0, 12, 24, 36, and 48 h. After biodegradation, 1 mL of samples was collected using a syringe and immediately used for protein extraction, and the residue was stored at –20°C for further chemical analyses and DNA extraction. The control reactor contained only rumen fluid or rice straw powder with sterile purified water instead of the rumen fluid. All incubations were performed in duplicate.2 and CH4) were measured as previously described  with a SHINCARBON-ST 50/80 packed column . Argon was used as the carrier gas, and injection and detection temperatures were set to 100 and 120°C, respectively. Carboxymethyl cellulase (CMCase) and xylanase activities were measured as previously described , total solids, VFAs, pH, and gas production (CObarcode for the prokaryotic 16S rRNA V4 region ((Illumina) by Seibutsu Giken. Sequence processing and taxonomic analyses were performed using QIIME2 ver. 2020.8  according to the manufacturer’s instructions. DNA libraries were constructed for amplicon sequencing based on two-step tailed PCR using primers with a 4 region . The con. 2020.8 , sequencSequence data are available in the DNA Data Bank of Japan (DDBJ) sequence read archive under accession number DRA014579 (DRR396149–DRR396178).g at 4°C for 10 min. The precipitate was washed with 1 mL of wash buffer  at 4°C and mixed with 1 mL of 2× sample buffer , containing 2 mmol L–1 phenylmethylsulfonyl fluoride and 1× proteinase inhibitor. Protein extraction was performed at 4°C using two rounds of 2 min bead-beating with Lysing Matrix E tubes , followed by centrifugation at 12,000×g at 4°C for 10 min. The supernatant was collected and stored at –80°C until SDS-PAGE and zymogram analyses.One milliliter of rumen fluid was centrifuged at 12,000×phosphate citrate buffer  containing 1.5% β-cyclodextrin for 30 min. CMCase and xylanase zymograms were obtained at 37°C for 1.5 and 4 h, respectively, in 30 mM sodium acetate buffer. Gels were stained with 0.1% Congo red, and band densities were analyzed using Fiji software (SDS-PAGE and zymography were performed as previously described (–1), and this increase was rapid during the first 12 h (from 85.4 to 119.0 mmol L–1). Due to the accumulation of VFAs, pH declined from 6.8 to 5.3  were lower than those in the biodegradation of rice straw powder with rumen fluid  over 48 h. At the genus level, the relative abundance of Prevotella decreased by 13.06% over 48 h  and decreased to 0.00%, which was similar to the change observed in 51-kDa CMCase activity.The structure of the fungal community also changed over 12 h and was clustered at 24, 36, and 48 h B. At theecomyces . The relEntodinium was the most dominant, followed by Diplodinium, Isotricha, Dasytricha, Polyplastron, and Eremoplastron  and the surface structure of polysaccharides.We also investigated changes in fibrolytic isozyme activities for 24 h when cellulose and hemicellulose were efficiently biodegraded. The band density of 23-kDa xylanase was the highest at 0 h, but decreased as biodegradation proceeded. Similarly, 51 and 140 kDa CMCases and 57 kDa xylanase exhibited decreased activities after 12 h. The band densities of 37, 53, and 58 kDa CMCases and 38, 44, and 130 kDa xylanases increased after 24 h. These results suggest that 51- and 140-kDa CMCases and 23- and 57-kDa xylanases play important roles in the early hydrolysis of carbohydrate polymers (0–12 h), whereas 37-, 53-, and 58-kDa CMCases and 38-, 44-, and 130-kDa xylanases are important for late hydrolysis (24–36 h). Changes in fibrolytic isozyme activity over time were also observed in our previous study, which evaluated the biodegradation of tomato leaves with rumen fluid  and Cyllamyces (36%)  from the rumen contents of mono-faunated sheep and found that P. multivesiculatum had the highest CMCase activity, producing 28- to 116-kDa CMCases. Previous metatranscriptomic analyses of the rumen microbiomes of adult Holstein cows revealed that Polyplastron is the major cellulolytic protozoa, contributing to 20% of protozoan cellulase transcripts , fungi , and protozoa (Dasytricha and Polyplastron). Nine of the ten major fibrolytic isozymes were related to microbial abundance in this study; however, a relationship was not observed between relative microbial abundance and 140 kDa CMCase activity. Previous 16S rRNA-based studies reported that approximately 77% of fiber-associated rumen microorganisms were uncultured , fungi , and protozoa (Dasytricha and Polyplastron).and  The present results will provide a foundation for elucidating the relationships between microbial and enzymatic characteristics during the biodegradation of lignocellulosic biomass.Overall, our chemical and zymogram analyses revealed that 37-, 51-, 53-, 58-, and 140-kDa CMCases and 23-, 38-, 44-, 57-, and 130-kDa xylanases had high band densities during rice straw powder hydrolysis. The microbial analysis showed that fibrolytic isozyme activity was related to the relative abundances of the dominant bacteria  Relationship Between Rumen Microbial Composition and Fibrolytic Isozyme Activity During the Biodegradation of Rice Straw Powder Using Rumen Fluid. https://doi.org/10.1264/jsme2.ME23041Supplementary Material


Pseudomonas aeruginosa is a major human pathogen, particularly effective at colonizing the airways of patients with cystic fibrosis. Bacteriophages are highly abundant at infection sites, but their impact on mammalian immunity remains unclear. We previously showed that Pf4, a temperate filamentous bacteriophage produced by P. aeruginosa, modifies the innate immune response to P. aeruginosa infections via TLR3 signaling, but the underlying mechanisms remained unclear. Notably, Pf4 is a single-stranded DNA and lysogenic phage, and its production does not typically result in lysis of its bacterial host. We identified previously that internalization of Pf4 by human or murine immune cells triggers maladaptive viral pattern recognition receptors and resulted in bacterial persistence based on the presence of phage RNA. We report now that Pf4 phage dampens inflammatory responses to bacterial endotoxin and that this is mediated in part via bacterial vesicles attached to phage particles. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and play a key role in host pathogen interaction. Recently, evidence has emerged that OMVs differentially package small RNAs. In this study, we show that Pf4 are decorated with OMVs that remain affixed to Pf4 despite of purification steps. These phages are endocytosed by human cells and delivered to endosomal vesicles. We demonstrate that short RNAs within the OMVs form hairpin structures that trigger TLR3-dependent type I interferon production and antagonize production of antibacterial cytokines and chemokines. In particular, Pf4 phages inhibit CXCL5, preventing efficient neutrophil chemotaxis in response to endotoxin. Moreover, blocking IFNAR or TLR3 signaling abrogates the effect of Pf4 bound to OMVs on macrophage activation. In a murine acute pneumonia model, mice treated with Pf4 associated with OMVs show significantly less neutrophil infiltration in BAL fluid than mice treated with purified Pf4. These changes in macrophage phenotype are functionally relevant: conditioned media from cells exposed to Pf4 decorated with OMVs are significantly less effective at inducing neutrophil migration in vitro and in vivo. These results suggest that Pf4 phages alter innate immunity to bacterial endotoxin and OMVs, potentially dampening inflammation at sites of bacterial colonization or infection. Graphical abstract—a model for OMV/Pf4 complex-mediated modification of the myeloid cell response to bacterial stimulation. While extruding from its host P. aeruginosa, Pf4 gets associated with OMVs. OMV/Pf4 complexes are endocytosed by lung-resident myeloid cells such as macrophages. Degradation-protected hairpin-structured RNA carried within OMVs triggers TLR3 activation. TLR3 signaling induces type I IFN production which in turn inhibits pro-inflammatory cytokine production in response to other bacterial products. Among the cytokines inhibited are neutrophil chemoattractants such as CXCL1 and CXCL5 resulting in abrogated neutrophil influx to the site of P. aeruginosa infection and eventually to the persistence of the bacteria. This bacterium has evolved several mechanisms to evade host immune responses, including the formation of biofilms, modifications of lipopolysaccharides (LPS), production of virulence factors, antibiotic tolerance, and secretion of exopolysaccharides. These mechanisms result in persistent bacterial infections, and their understanding is critical for development of effective therapies against P. aeruginosa infections.P. aeruginosa. However, it remains unclear how the innate immune system senses phages or how phages impact sensing of bacteria in the human host  on their surface. LPS molecules present on the surface of OMVs can activate TLR4 on host immune cells, triggering pro-inflammatory cytokine production, e.g., TNF-α or the neutrophil chemoattractant CXCL5 that facilitate bacterial clearance  attached to Pf4 phages in these effects. OMVs are small, spherical proteoliposomes (200–400 nm diameter) that are shed from the outer membrane of Gram-negative bacteria, particularly at time of stress or bacterial lysis by phages, including in learance .P. aeruginosa infections.Our findings provide new insights into the interactions between filamentous phages, OMVs, and host innate immune cells which have important implications for the development of novel therapeutic strategies against 22.1P. aeruginosa isolate mPAO1 served as a wild type that harbors the filamentous phage Pf4 in all experiments , or Luria-Bertani  at 37°C.The eriments . Isogenieriments . Unless 2.2P. aeruginosa mPAO1 was used to produce Pf4. Mock phage preparations from PAO1ΔPf4 were prepared according to the same protocol as other phage samples. Cultures were inoculated with an OD600 of 0.1 and grown to the mid-log phase in which the respective phage was added and co-incubated overnight. The next day, the bacteria were harvested at 6,000 × g; 30 min; 4°C. The supernatant was sterile filtered using a 0.22-µm bottle top filter and treated with 50 U/ml of benzonase nuclease  or RNase-free DNase I nuclease  at 37°C for 2 h. An aliquot of the phage solution was left untreated (impure phage lysate). The remaining phage solution was incubated with polyethylene glycol 8000  at 4°C overnight either once (1× PEG) or twice (2× PEG) as described previously , 100 nM coaB primer, and 2 µl supernatant, 1× PEG or 2× PEG Pf4 preparation. Cycling conditions were set to the following: 50°C 2 min; 95°C 2 min;  × 40 cycles. A standard curve was constructed using a plasmid containing the template sequence at a known copy number per ml. Pf4 copy numbers were then calculated by fitting Ct values of the unknown samples to the standard curve.Pf4 virion copy number was measured using qRT-PCR as previously described . Briefly2.3600 greater than 4. The cultures were separated by centrifugation at 6,000 × g for 60 min at 4°C. The supernatant was sterile filtered using a 0.22-µm bottle top filter to remove any intact cells. To ensure that no bacteria remained in the filtered supernatant, a 1-ml aliquot was plated onto LB agar plates and incubated at 37°C overnight. The OMVs in the supernatant were harvested by ultracentrifugation 150,000 × g for 4 h at 4°C as previously described . Labeled OMVs and Pf4 were stored in 1 × PBS at 4°C.2× PEG-treated Pf4 lysate was labeled with Alexa Fluor 488 TFP ester , and OMVs were labeled with either DiD′ , SYTO RNASelect , and BODIPY  according to the manufacturer’s instructions. Unincorporated dye was removed by three ultracentrifugation rounds  were then co-incubated with RAW 264.7 at 37°C and 5% CO2 for 1 h. Subsequently, cells were fixed, permeabilized at room temperature for 20 min, stained with phalloidin-CF555 conjugate, and incubated with 1 µg/ml DAPI at room temperature for 10 min. The cover slip was transferred onto a glass slide using ProLong Gold Mountant (Invitrogen Cat. No. P36930), and confocal microscopy was performed.1.5 × 102.65 U937 macrophages were seeded per well into a 24-well plate and were either left untreated or treated with 1011 pfu/ml AlexaFluor488-labeled Pf4 with or without 1 μg/ml DiD-labeled OMV, or RNASelect-labeled OMV (by protein) at 37°C and 5% CO2 for 1.5 h. When uptake inhibitor treatments were used, cells were pretreated with 100 ng/ml wortmannin , 1 µg/ml nocodazole , 5 µM chlorpromazine , and 2 µM cytochalasin D . Samples were stained with Zombie Near-IR Live-Dead dye  and run live on a Cytek Northern Lights 3000 flow cytometer. Data were analyzed on FlowJo™ software.2.5 × 102.7Human U937 macrophages  were maintained in RPMI  supplemented with 10% FBS , penicillin–streptomycin , and sodium pyruvate . Murine RAW 264.7  were maintained in DMEM  supplemented with 10% FBS , penicillin–streptomycin , and sodium pyruvate . HEK-Blue hTLR3  and HEK-Blue IFNα/β  were maintained in DMEM as above, with the addition of 30 µg/ml of blasticidin  and 100 µg/ml of Zeocin™  as per the manufacturer’s instructions.2.7.14 cells/well in a 96-well plate. Unless otherwise specified, stimuli were as follows: 1 × 108 pfu/well of purified phage preparation, 10 ng/ml purified OMVs, 100 ng/ml LPS , and 10 μg/ml high molecular weight poly (I:C) . The TLR3 signaling inhibitor  was used at 27 μM. Cells were incubated at 37°C and 5% CO2 for the indicated timepoints and then centrifuged at 300 × g for 5 min prior to supernatant removal.For cytokine release and reporter assays, cells were seeded at a density of 5 × 102.7.2Human CXCL5 , human TNF-α , human IFNβ , mouse CXCL1 , mouse CXCL5 , and mouse TNFα  were performed using the manufacturer’s instructions. Absorbance was read on a Spark microplate reader (Tecan).2.7.3Cytotoxicity was determined using the LDH-Glo kit  as per the manufacturer’s instructions. Absorbance was read on a Spark microplate reader (Tecan).2.7.44 cells/well. Cells were stimulated for 24 h with 1 × 109 pfu/well of purified Pf4 phage or equivalent volume ΔPf4 preparation, 10 ng/ml purified OMVs, and 100 ng/ml LPS . Cells were centrifuged at 300 × g for 5 min, and supernatant was collected and used for cytokine profiling through Luminex . Human 89-plex kits were purchased from eBioscience/Affymetrix and used accordingly to the manufacturer’s recommendations with modifications as described below. Briefly, beads were added to a 96-well plate and washed in a BioTek ELx405 washer. Samples were added to the plate containing the mixed antibody-linked beads and incubated at room temperature for 1 h followed by overnight incubation at 4°C with shaking. Cold- and room-temperature incubation steps were performed on an orbital shaker at 500–600 rpm. Following the overnight incubation, plates were washed in a BioTek ELx405 washer and then biotinylated detection antibody added for 75 min at room temperature with shaking. The plate was washed as described above, and streptavidin-PE was added. After incubation for 30 min at room temperature, washing was performed as above and the reading buffer was added to the wells. Each sample was measured in duplicate. Plates were read using a Luminex 200 instrument with a lower bound of 50 beads per sample per cytokine. Custom assay Control beads by Radix Biosolutions were added to all wells.U937 macrophages were plated in 96-well plates at 5 × 102.7.4.12 fold change with respect to the control LPS sample. Data were plotted as a heatmap using the heatmap package (v1.0.12) in R.Technical replicate MFI values were averages for each sample and transformed into log2.8in situ by coherent anti-Stokes Raman scattering (CARS) microscopy with an inverted microscope . The vibrations were coherently driven by two laser beams overlapped in time and space, generated by a ps-pulsed laser system  with a 1,031-nm Nd:VAN laser and an optical parametric oscillator (OPO), tunable between 700 and 960 nm . The OPO wavelength was set to 797 nm to address the lipid-specific symmetric stretching vibration of CH2 at 2,848 cm−1. The quadratic dependence of the CARS signal on the number density of the probed vibrational group provides sharp contrast for the lipid-dense regions without the need for external labels. The excitation powers at the sample were 13 mW for the 1,031-nm beam and 27 mW for the OPO beam, and CARS signals were collected in the forward direction with a photomultiplier tube . Images with multiple phages within the field of view were captured in a single focal plane with 10.8-μs dwell time and 20-nm pixel size. The acquired images were prepared and analyzed in ImageJ. After east shadow correction, the images were bandpass Fourier filtered between 2 and 100 pixels with 5% tolerance of direction and suppression of horizontal stripes. A Gaussian blur filter (sigma = 1.5) was applied, and the background was subtracted by a rolling ball (radius 80 pixels). For spectral acquisitions, Z-stacks (15 slices) were collected on individual phages (n = 4) with 5.3-μs dwell time, 12-nm pixel size, and 0.2-μm vertical separation between adjacent z-positions. The stacks were east shadow corrected, and the total intensity for a region of interest (ROI) manually created around the phage was evaluated for a range of vibrations (with the OPO tuned from 800 to 785.5 nm in steps of 0.5 nm) in the C-Hx stretching region. The background signal from water was evaluated in ROIs of similar areas a distance away from the phage. The background had no discernable peaks and was subtracted from the spectrum. On the same microscope platform, equipped with a LU-N4 excitation source and a C2-DUVB GaAsP detector unit, the fluorescence from phages labeled with AF514 were collected with an excitation wavelength of 488 nm and 10.8-µs dwell time and 20-nm pixel size. The confocal fluorescence images were processed in ImageJ with a smoothing function and a rolling ball (radius 80 pixels) background subtraction.1× PEG Pf4 phages in PBS were adsorbed onto coverslips and characterized 2.95 µl of phage lysates and OMV preparations (~50 µg/ml protein) were absorbed onto glow discharged formvar and carbon-coated copper grids  at room temperature for 5 min, negatively stained with 1% (w/v) uranyl acetate at room temperature for 1 min, and imaged with a JEM-1400 transmission electron microscope (JEOL) operated at 120 kV. The images were taken with a Gatan Multiscan 791 CCD camera. Immunogold labeling of OMV was performed using 5 µl of 1× PEG-treated Pf4 lysates (~50 µg/ml protein) that were absorbed onto formvar- and carbon-coated copper grids at room temperature for 5 min and subsequently blocked in 2.5% goat serum  at room temperature for 15 min. The samples were incubated in α-OprF rabbit serum  at room temperature for 15 min, followed by an incubation in goat-anti-rabbit IgG serum conjugated to 10-nm gold particles  for 15 min and negative staining in 1% (w/v) uranyl acetate for 1 min.2.10RNA was extracted from OMV preparations isolated from cultures of PAO1 or PAO1 ΔPf4. Each preparation was split into three aliquots which were incubated at 37°C for 1 h with either RNase free DNase I , proteinase K , and RNase free DNase I or left untreated. OMVs were disrupted by adding 3% (w/v) SDS . The hot phenol extraction protocol initially published by 2.11After RNA extraction from OMVs, the samples were depleted from rRNA using the RiboMinus™ Bacteria 2.0 Transcriptome Isolation Kit . The samples were further enriched for small RNA species (<200 nt) using the RNA Clean and Concentrator Kit . Sequencing libraries were prepared with the KAPA Total RNA Kit for Stranded RNA HyperPrep  and sequenced by on the Illumina platform .Quality control was performed on raw data using FASTQC 0.11.9 and MULTIQC . Adapter2.12600 of 1) were pelleted by centrifugation  from the respective P. aeruginosa culture. Cell pellets were resuspended in 60 µl of 5× sample buffer according to Laemmli, boiled for 10 min at 100°C, and either used for SDS-PAGE or stored at −20°C (For whole-cell lysate (WCL) preparation, equal amounts of cells  was used as a molecular mass standard. Subsequently, protein bands were stained with Coomassie solution  1:20,000 diluted in casein-BSA buffer for 30 min at room temperature. Reactive protein bands were detected by using the LI-COR Odyssey 9120 imaging system.Proteins contained in WCL or OMVs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using SurePAGE™, Bis-Tris , and MES SDS running buffer (GenScript. Cat. No. M00677). The Chameleonsolution  or furth2.13x g for 30 min at 22°C with no brake, and neutrophils from the interface of the Histopaque-1119 and Histopaque-1077 layers were collected and washed in RPMI + 10% FBS. Count and viability of neutrophils was determined by trypan blue exclusion.Human neutrophil isolation protocol was adapted from Swamydas et al. . Briefly2.14P. aeruginosa PAO1 or PAO1ΔPf4 was grown to the mid-exponential phase  in 4 ml LB broth at 37°C with aeration. The PAO1 culture was infected with 100 µl of Pf4 stock (1010 pfu/ml) or PBS control to promote phage production. These cultures were grown overnight at 37°C with aeration. On the following day, bacteria were pelleted by centrifugation at 6,000 × g for 10 min, washed three times in sterile PBS, and resuspended in PBS to a final concentration of 3 × 108 CFU/ml. Mice  were intranasally inoculated with 1.5 × 107 CFU/ml in 50 µl PBS of each strain or PBS control.Experiments were performed as described in Secor et al.. Briefly9 pfu/ml of 1× PEG or 2× PEG Pf4, 10 ng/ml OMVs, or 10 ng/ml LPS were prepared in sterile PBS. Mice were intranasally inoculated with 50 µl of the respective inoculum.For the inocula using isolated phages and/or OMVs, either 10+ gate was reported, as well as live neutrophils within the CD45+ Ly6G+ gate. Absolute cell counts were adjusted by total BAL volume recovered.Mice were sacrificed at 24 h post infection. The lungs were lavaged with 0.8 ml sterile PBS as described previously . BAL flu2.15p < 0.05. Non-significance was indicated by the letters “n.s.”. For experiments involving cell assays, each replicate was normalized against a specified control condition to control for varying inter-experiment intensities of cytokine production or reporter protein secretion.Where n is not stated, graphs show a representative experiment of n ≥ 3 assays, with n ≥ 3 technical or biological replicates. All statistical analyses were performed using GraphPad Prism . Significance was assessed by ANOVA followed by Holm–Šídák test for multiple comparisons unless otherwise indicated. Depicted are means with standard deviation (SD) of the replicates unless otherwise stated. Statistical significance was considered 33.1Since Pf phages are ssDNA viruses and TLR3 is a sensor for dsRNA, it was unclear how these responses are mediated . Given tTo test this, we evaluated the impact of performing one versus two rounds of polyethylene glycol (PEG) precipitation on our phage preparations (P. aeruginosa which tethers the outer membrane to the peptidoglycan layer and regulates OMV production (Transmission electron microscopy (TEM) was used to examine isolated Pf4 phages. Spherical structures were observed on the distal ends of Pf4 , includioduction . The preA second orthogonal imaging approach, coherent anti-Stokes Raman scattering (CARS) microscopy for label-free visualization of lipids, was used to confirm the presence of lipid deposits at the end of Pf4 particles. CARS is a microscopy tool for label-free visualization of lipid bilayers that could be expected to reveal the presence of OMVs. CARS imaging of 1× PEG Pf4 indeed indicated the presence of large multilayer lipid droplets on the distal ends of the filamentous phage particles  mimic the effects of 1× PEG Pf4 preparations. To this end, a Luminex assay was performed on supernatants derived from human U937 macrophages incubated for 24 h with various combinations of LPS, 1× PEG Pf4, OMVs, and 2× PEG Pf4 with OMVs . ConversWe then assessed the effects of OMV-supplemented Pf4 on CXCL5 production and the dependency of this present system on TLR3 by treating human U937 macrophages with a small-molecule compound that specifically prevents dsRNAs from binding to TLR3  and founTo establish that Pf4/OMV complexes induce type I IFN production, we employed a HEF-IFNα/β cell line which reports on engagement of the IFNAR1/2 receptor. These HEK-IFNα/β cells were incubated with conditioned media from human U937 macrophages stimulated with various reagents for 24 h, and the degree of IFNAR stimulation was considered to be a proxy for IFNα/β concentration in the conditioned media. While 2× PEG Pf4, mock preparation, and OMVs did not induce appreciable type I IFN production above the LPS control, cells co-stimulated with LPS together with 2× PEG Pf4/OMV produced the biggest IFN response  fluid and quantified the cell influx into the lung with flow cytometry, as well as the chemokine levels with ELISA  can be found below: NCBI, PRJNA994600.Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. The animal study was approved by Stanford University Animal Welfare Board/IACUC. The study was conducted in accordance with the local legislation and institutional requirements.NP, MP, and PB contributed to conception, design, and supervision of the study. NP, MP, PB, GK, and AE were responsible for methodology. NP, MP, FD, NH, AH, PJ, AE, and GK were involved in the investigation and experimental performance. NP and MP wrote all sections of the manuscript. All authors contributed to the article and approved the submitted version.


Solanum lycopersicum βCA1 (SlβCA1) was significantly upregulated in plants treated with Xanthomonas euvesicatoria 85-10. The protein was localized in the nucleus, cell membrane and chloroplast. Using tomato plants silenced with SlβCA1, we demonstrated that SlβCA1 plays an active role in plant disease resistance. Moreover, we found that the elicitor PopW upregulated the expression of SlβCA1, while the microbe-associated molecular pattern response induced by PopW was inhibited in TRV-SlβCA1. The interaction between PopW and SlβCA1 was confirmed. Here, we found that SlβCA1 was positively regulated during PopW-induced resistance to Xanthomonas euvesicatoria 85-10. These data indicate the importance of SlβCA1 in plant basic immunity and its recognition by the Harpin protein PopW as a new target for elicitor recognition.β-Carbonic anhydrase (βCA) is very important for plant growth and development, but its function in immunity has also been examined. In this study, we found that the expression level of  Plant diseases are considered one of the most important factors threatening plant growth and yield . Plants 2-binding protein that enzymatically catalyzes the interconversion of CO2 and HCO3− [Carbonic anhydrase (CA), a zinc-containing metalloenzyme, is a COnd HCO3− ,5. In annd HCO3− ,7. The Cnd HCO3− . During nd HCO3− . The orgnd HCO3− .βCA was inhibited after Phytophthora infestans infection [βCA gene was suppressed by infection with Plasmopara viticola [Hyaloperonospora parasitica by proteomic analysis [Nicotiana benthamiana, the suppression of βCA expression by genetic manipulation increases host susceptibility to P. infestans [Pseudomonas syringae pv. tomato (Pst) DC3000 [Pst DC3000 [N. benthamiana, βCA1 can inhibit the leaf-hypersensitive response induced by avrPto [The relationship between the function of βCA and photosynthesis is a major research direction . Severalnfection . Additioviticola . In contanalysis . Moreovenfestans . In Arab) DC3000 . Althougt DC3000 . In addiy avrPto . Salicyly avrPto , binds ty avrPto .βca1βca4 double mutant and the βca1βca4βca6 triple mutant were not sensitive to the changes in stomatal conductance induced by CO2 [Pst DC3000 was not damaged in the βca1βca2βca3βca4βca6 mutant, but effector-triggered immunity was impaired in the quintuple mutant [Pst DC3000, and the regulatory pathway is independent of stomatal and SA-dependent regulation [Although there is much evidence showing that βCA plays an important role in plant immunity, research on the specific resistance mechanism and molecular interaction mechanism of βCA is still in the early stage. A study showed that the d by CO2 . Hu et ad by CO2 . Notablyd by CO2 . The Arae mutant . Recentle mutant . Wang ete mutant . Howevergulation . The immRalstonia solanacearum (Rs) ZJ3721 [Xanthomonas euvesicatoria (Xe) 85-10 infection, and the resistance mechanism was related to stomatal immunity [PopW, as a biological elicitor of the Harpin protein, was originally isolated from ) ZJ3721 . PopW ca) ZJ3721 ,23,24. Iimmunity ,23,25.SlβCA1 gene after Xe 85-10 inoculation, we chose to study the function in basal immunity of SlβCA1 in the model plant tomato. We provide evidence that inhibition of SlβCA1 gene expression attenuates plant defense responses to Xe 85-10 infection. In addition, it was previously reported that βCA could act as a negative regulator of the elicitor-induced microbe-associated molecular pattern (MAMP) response. Therefore, we selected an elicitor, PopW, which can enhance tomato defense against Xe 85-10, to determine whether SlβCA1 can affect the MAMP response. The results showed that SlβCA1 can upregulate the PopW-induced MAMP response and directly interact with PopW, thus reducing PopW-induced resistance to Xe 85-10. The immune function of SlβCA1 is not related to CA activity. These findings provide insights into the role of βCA and PopW in plant immunity.Given the response of the βCAs exhibits different responses to infection by various pathogens in different plants [AtβCAs can respond to pathogen infection, among which AtβCA1, AtβCA2, AtβCA4 and AtβCA5 generally show reduced expression in response to infection with 16 plant pathogens, while AtβCA3 is significantly upregulated after treatment with P. syringae, Alternaria brassicicola, Rhizoctonia solani, Sclerotinia sclerotiorum and Golovinomyces cichoracarum, and AtCA6 is significantly upregulated after infection with P. infestans and Myzus persicae [SlβCA3 showed high expression after Pst DC3000 treatment [SlβCA3 after Pst DC3000 and Rs ZJ3721 treatment -SlβCA1 in membrane A.Amino acid sequence alignment of the tomato, Arabidopsis and tobacco proteins revealed a close relationship between SlβCA1 and NbβCA1.2 B. These SlβCA1 in tomato (TRV-SlβCA1) and used tomato with glucuronidase silenced (TRV-GUS) as a negative control  derived from Rs ZJ3721. As shown, after PopW treatment, SlβCA1 expression was significantly induced at 6 h , the accumulation of ROS and the accumulation of callose. Silencing of nescence B. The acreatment C. Moreov reduced D. Thus, N. benthamiana  assay to verify the interaction and found that SlβCA1 interacted with PopW A. In thethamiana C. Subseqthamiana D. In conNext, to explore their interacting domains, we divided PopW into the N-terminal with a Harpin domain and the C-terminal pectate lyase (PL) domain, while SlβCA1 was divided into the C-terminus with a CA domain and the N-terminus. The interaction between the N-terminus of PopW with the Harpin domain and the N-terminus of SlβCA1 without the CA domain was demonstrated by the Y2H and LCA methods A,C.SlβCA1 was less than that in TRV-GUS  reaction of Rubisco in C3 photosynthesis [Xe 85-10 treatment and to increased inhibition of pathogen growth , and the culture medium for tobacco was vermiculite.Tomato and https://sg.idtdna.com/Primerquest/Home/Index, accessed on 11 May 2022), SlβCA1-silenced fragments were selected from a tomato cDNA library. The fragment was cloned into the vector pTRV2 and transformed into Agrobacterium tumefaciens (GV3101) to silence the SlβCA1 gene. Specific procedures were carried out by Agrobacterium-mediated transformation [The downregulation of gene expression in tomato was achieved by virus-induced gene silencing. First, as described online  medium containing 50 μg/mL kanamycin at 28 °C. Additional pathogens, including Pst DC3000 and Rs ZJ3721, were cultured in LB medium; Pst DC3000 was cultured at 28 °C, and Rs ZJ3721 was cultured at 37 °C. For pathogen treatment, the bacterial suspension cultured overnight was resuspended in pure water to OD600 = 0.3, and 0.02% Silwet L 77 was added, after which the mixture was sprayed on the leaves of 8-week-old tomato plants [Extraction and purification of PopW were carried out as described earlier . Xe 85-1o plants .1, 102, 103, 104, 105, 106 and 107) and mixed well. Then, 10 μL was used for spot plating, colonies were counted after 4 d, and the colony-forming unit (CFU) number was calculated [Disease status was assessed by quantifying bacterial leaf populations . Bacterilculated .Amino acid sequence alignment and tree construction were performed using the neighbor-joining method with MEGA-X. The SlβCA1 protein is highlighted in red. The sequences included SlβCA1 (Solyc02g086820), SlβCA2 (Solyc05g005490), SlβCA3 (Solyc02g067750), SlβCA4 (Solyc09g010970), NbβCA1.1 (Niben101Scf09253g00011.1), NbβCA1.2 (Niben101Scf06349g00008.1), NbβCA2.1 (Niben101Scf08637g01001.1), NbβCA2.2 (Niben101Scf01155g01009.1), NbβCA3.1 (Niben101Scf16378g01012.1), NbβCA3.2 (Niben101Scf03766g09001.1), NbβCA4.1 (Niben101Scf07739g00010.1), NbβCA4.2 (Niben101Scf13995g01012.1), AtβCA1 (AT3G01500), AtβCA2 (AT5G14740), AtβCA3 (AT1G23730), AtβCA4 (AT1G70410), AtβCA5 (AT4G33580) and AtβCA6 (AT1G58180).SlUBI3 as an internal control [CAs and resistance marker genes were determined.Measurement of gene expression was performed by using real-time fluorescence quantitative PCR with  control . Total RXe 85-10 or PopW.The production of ROS was monitored in real time by chemiluminescence within half an hour after treatment , while DXe 85-10 for 12 h were selected for the experiment and examined under an inverted microscope after decolorization.The staining method for callogens detection was performed as described by Yuan et al. . Tomato The determination of protein interactions was performed by four methods, including a BIFC assay, COIP of plant protein extracts, a Y2H assay and an LCA. The specific methods were as follows:Agrobacterium transformation, please refer to the method of Seo et al. [Agrobacterium carrying pCAMBIA1300-SlβCA1 and pCAMBIA2300-PopW was injected into tobacco leaves for 48 h, and total protein was extracted with lysate (Beyotime). For the next IP run, we used Flag beads to enrich Flag protein complexes (Beyotime). Detection of the total protein content (Input) and Flag protein complex (Output) was performed with GFP and Flag .In the COIP experiment, SlβCA1 was cloned into the pCAMBIA1300 vector with a GFP tag, and PopW was cloned into the pCAMBIA2300 vector with a Flag tag . The pCAo et al. . AgrobacIn the Y2H experiment, pBT3-SUC and pPR3 were used as bait and prey vectors, respectively . The conA. tumefaciens strains carrying the pCV-C-PopW and pCV-N-SlβCA1 vectors were coinjected into tobacco. After 48 h, the tobacco leaves were placed under a laser confocal scanning microscope to observe the fluorescence status. If the target proteins interacted, the fluorescence signal of YFP appeared in the field of vision.In the BIFC experiment, pCV-C/N was used as the vector, with the C-terminus linked with PopW and the N-terminus linked with SlβCA1 . A. tumeLCA was performed using the method of Zhou et al. . We consAgrobacterium. We used DAPI staining as a marker for the localization of the nucleus and detected the autofluorescence of the chloroplast as a marker. The fluorescence of the final GFP fusion protein, DAPI fluorescence and autofluorescence of chlorophyll were observed together in tobacco leaves using a laser functional confocal microscope .The specific method used for subcellular localization was as described by Zhang et al. . In brieXe 85-10, approximately 0.1 g of fresh leaf tissue was ground in liquid nitrogen, and 1 mL of extraction buffer , pH = 7.4) was added. The sample was centrifuged at 8000 rpm for 30 min, and the supernatant was examined. The determination of CA enzyme activity was performed with a Plant βCA Enzyme-Linked Immunosorbent Assay (ELISA) Kit .According to a previously described method by Hatch et al., CA was extracted from leaves . ImmediaXe 85-10 for 3 h, epidermal tissues were removed with tweezers and placed under an inverted microscope to observe the stomatal apertures. Visual fields were randomly selected to measure the stomatal apertures, and at least n ≥ 100 stomata were measured in each treatment.The method used for the determination of porosity was as previously described by Melotto et al. with slight modifications . The metp < 0.05; ** indicates p < 0.01). Orign 2018 was used for data processing and mapping.All experiments were performed in triplicate, and the values in the figures represent the mean ± SD of three replicates . AsterisSlβCA1 could respond to the treatment with the tomato scab pathogen Xe 85-10, so SlβCA1 may participate in the immune response of tomato to Xe 85-10. First, we observed that the Xe 85-10 colonization level in TRV-SlβCA1 increased, and the expression of resistance genes was inhibited. In addition, the expression of SlβCA1 was also induced by PopW. We systematically analyzed the PopW-induced MAMP response and found that SlβCA1 could inhibit PopW-induced ROS burst and callose accumulation. PopW and SlβCA1 could directly interact. The disease resistance of tomato against Xe 85-10 induced by PopW was affected, including via ROS burst, the accumulation of callose, reduction in the colonization level, upregulation of disease-resistance genes and closure of stomata. The CA activity of SlβCA itself was not involved in these immune processes. Our data highlight the important role played by SlβCA1 in basic plant immunity, supporting further research on the function of CA in immunity. The mechanism by which CA participates in surface recognition and interacts with PopW during in vivo interactions between plants and pathogens and, crucially, the identification of CA as a plant cell surface receptor were reported here for the first time, providing novel insights into the direction of the interactions between plants and pathogens.In this study, SlβCA1 was the main target and was found to be localized in the cell membrane, nucleus and chloroplast.