Escherichia coli. PA at relatively high amount (40–100 μL) in a 30-mLmating system inhibited conjugation by 74–85%, following anorder of PA > F3 ≈ F2 ≈ F1, proving the hypothesisthatPA amendments may mitigate soil ARG pollution by inhibiting HGT. Thebacteriostasis caused by antibacterial components of PA, includingacids, phenols, and alcohols, as well as its acidity (pH 2.81) contributedto the inhibited conjugation. However, a relatively low amount (10–20μL) of PA in the same mating system enhanced ARG transfer by26–47%, following an order of PA > F3 ≈ F2 > F1.Theopposite effect at low amount is mainly attributed to the increasedintracellular reactive oxygen species production, enhanced cell membranepermeability, increased extracellular polymeric substance contents,and reduced cell surface charge. Our findings highlight the hormesis(low-amount promotion and high-amount inhibition) of PA amendmentson ARG conjugation and provide evidence for selecting an appropriateamount of PA amendment to control the dissemination of soil ARGs.Moreover, the promoted conjugation also triggers questions regardingthe potential risks of soil amendments  in the spread ofARGs via HGT.Spread of antibioticresistance genes (ARGs) by conjugationposesgreat challenges to public health. Application of pyroligneous acids(PA) as soil amendments has been evidenced as a practical strategyto remediate pollution of ARGs in soils. However, little is knownabout PA effects on horizontal gene transfer (HGT) of ARGs by conjugation.This study investigated the effects of a woody waste-derived PA preparedat 450°C and its three distillation components at different temperatures  on conjugativetransfer of plasmid RP4 within  AMR would cause an annual death of approximately10 million by 2050 and over US$100 trillion economic burden over thenext few decades without effective measure.17 inactivation of ARGsand antibiotic resistant bacteria from the reclaimed water beforeirrigation,18 vermiculture,20 phage therapy,21 and soil amendment  applications.23 Among them, soil amendments,23 which may fight against ARGs already existing in soils, have attractedincreasing attention because of their efficiencies and convenientimplementation.23 Pyroligneous acids (PAs)derived from biomass pyrolysis, as multifunctional soil amendmentswith long history, can decrease soil salinity and pH,25 regulate bacterial community,24 killplant pathogenic fungi and bacteria,28 and promote crop growthand grain yield.25 Our recent studies firstdemonstrated that PA amendment can reduce ARG levels in soils grownwith vegetable Brassica, regardlessindividual application or combined with biochar amendment.29 Similarly, another research also reported that the combined treatmentof bamboo biochar and distilled PA showed significant synergisticeffects on alleviating ARG proliferation in municipal solid wastecomposts.30 These studies mainly ascribedthe decreased ARG abundances to the inhibited HGT, reduced co-selectionof heavy metals and lowered potential host bacteria of ARGs but ignoredthe underlying mechanisms of PA on HGT process. Previous studies haveverified that PA as a bacteriostatic agent can damage the cell structureand inhibit the bacterial energy production and cell metabolic activity,28 which may impede the conjugative process. However, these potentialeffects and the relevant mechanisms underlying HGT process by PA amendmentremain unclear, limiting the further development of the PA-based technologyto abating soil ARG pollution.Increasing strategies are recommended to alleviate ARG pollutionin agricultural soils, such as aerobic composting or anaerobic digestionof manure and sludge before use,32 is commonlyknown to have a great impact on transferring ARGs among bacterialspecies.33 Particularly, conjugation, as a major pathwayof HGT,34 can disseminate ARGs carriedon mobile genetic elements such as plasmids, insertion sequences,and transposons32 via conjugation bridgesformed by a conjugative/sex pilus or membrane pores within intragenericor intergeneric bacteria.32 Increasing evidencedocumented that antibiotics 35 and non-antibiotic chemicals 37 at sub-inhibitory levels,that is, below the minimum inhibition concentration (MIC), can promoteARG conjugative transfer. The potential mechanisms include the overproducedreactive oxygen species (ROS), enhanced membrane permeability, promotedcell-to-cell contact by stimulating extracellular polymeric substance(EPS) production and decreasing electrostatic repulsion between cellsurfaces, induced SOS response, and regulated conjugation-relatedgene expression.38 On the contrary, these antibiotics39 and non-antibiotic chemicals40 at inhibitorylevels (above the MIC) generally inhibited ARG conjugation by strongbacteriostasis. As common soil amendments, PAs containing a largevariety of water-soluble compounds  suchas organic acids, phenolics, esters, aldehydes, and alcohols41 can effectively inactivate bacteria,26 fungi,28 plant pathogens,27 and viruses.42 Astudy reported that a PA obtained by slow pyrolysis of a mixture ofpine, spruce, and fir wood particles, containing organic acids suchas acetic acid and aldehydes like vanillin and furfural, all of whichare known as microbial inhibitors, showed antibacterial activity againstfive pathogenic bacterial strains.43 Also,a recent study reported that phenolic compounds , one typeof the representative components of PA, at 10–100 mg/L significantlypromoted conjugative transfer of plasmid RP4 from Escherichiacoli (E. coil) HB101 to the bacterial community inactivated sludge by increasing ROS levels and bacterial membrane permeability.44 Therefore, we hypothesized that PA amendmentcould induce hormesis on intrageneric conjugative transfer of ARGs,that is, inhibition at high amount by bacteriostasis via antimicrobialcomponents and acidity of PA but promotion at low amount by enhancingROS production, membrane permeability, and intercellular contact.HGT, including conjugation, transformation,transduction, gene-transferagents, and cell fusion in prokaryotes,E. coli. The specific objectives were to (1) investigatethe amount effect of PA on the intrageneric conjugation of ARGs, (2)evaluate the roles of its distilled fractions and representative componentsas well as PA acidity on the conjugation, and (3) explore the potentialmechanisms behind the conjugation regulated by PA. This study willexpand the insights into PA application as a feasible strategy forcontrolling the spread of soil ARGs.To test this hypothesis, the present study investigated the effectsof a pristine PA prepared by charring woody waste at 450°C andits three distillation components at different temperatures  on the conjugative ARG transfer within the genus 22.1E. coli strains(HB1011 and NK5449) were used tobuild an intrageneric conjugation model of ARGs mediated by transferableplasmid.29E. coli strains, belonging to the phylum Proteobacteria, were chosen because they are one type of the most abundant bacterialspecies in soils and other environments.45 They are also frequently used as the soil bacterial surrogates.38E. coli HB101 harboring transferable plasmid RP4, a typical plasmid namedIncP α-type, carries three ARGs against ampicillin , tetracycline , andkanamycin . E. coil NK5449 carries both the resistance genes for rifampicin  and nalidixic acid .Two 2.229 Three distilled fractions of the pristinePA were obtained by a conventional atmospheric distillation methodinvolving passive volatilization of compounds at 98 (F1), 130 (F2),and 220 (F3)°C as previously reported;29 these fractions are commonly used as antimicrobial agents and forsoil amendment.29 It takes more than 6 months tostabilize and relatively purify PA before use as in a previous study,29 which would exclude the effect of degradationproducts of PA and its fractions on conjugation. The properties ofpH, density, and chemical composition of the pristine PA and its fractionswere characterized previously,29 and theyare presented in the Supporting Information . Additionally, acetic acid (>99.5% pure), guaiacol(99%pure), 2,6-dimethoxyphenol (98% pure), and 3-methyl-1,2-cyclopentadione(98% pure), four representative components of the PA and the distilledfractions , were obtained fromSinopharm Chemical Reagent Co., Ltd., China, and Guangzhou ChemicalReagent Factory, China.PA was pyrolyzed from the blended woody wastes as our previousstudies.2.329 The modelwas established using E. coli HB101harboring transferable plasmid RP4 as the donor and E. coli NK5449 as the recipient in the intragenericconjugation model. Briefly, after being incubated in 100 mL LB brothmedium at 37°C for 12 h with an oscillating speed of 200 rpm,the donor E. coli HB101 and recipient E. coli NK5449 strains were centrifuged at 4000 rpmand 25°C for 10 min to remove the supernatants. The bacteriapellets were washed twice using phosphate buffer saline (pH 7.2) andthen resuspended in LB liquid medium to obtain the desired bacterialconcentration (3 × 108 CFU/mL). Subsequently, 150μL of donor and recipient bacteria suspensions was added into30 mL LB liquid medium containing different amounts of PA , the three distilled fractions , or four representative components includingacetic acid , 2-methoxy-phenolx , 2,6-dimethoxy phenol , or3-methyl-1,2-cyclopentanedione . Notably,the amounts of PA and its three distilled fractions were added accordingto the recommend agriculture-applied levels (Table S1) and their MICs to the donor and recipient strains (Table S2), and the added amounts of these fourcomponents were selected based on the corresponding contents of thesefour components in PA and its distilled fractions  and their MICs on the donor and recipient strains(Table S2). The nutrition-rich liquid LBsystem at 37°C may ensure the growth of bacterial donors, recipients,and transconjugants without any nutrient limitation, which could reflectthe HGT process in the agricultural soils with a high level of nutrientdue to extensive application of chemical fertilizers and manures.46 After being incubated at 37 °C and 200 rpm for 18 h, the mixtureswere appropriately diluted in NaCl (0.9%) and plated on LB-agar platescontaining the corresponding antibiotics to select the transconjugants. After the plates were cultivated at 37°C for 24 h, the transconjugants,donors, and recipients were counted. Then, the conjugative transferfrequency was calculated as the ratio of the transconjugant numberto the total recipient number in the control.14 Each treatment was set with biological triplicates in three 50 mLsterile conical bottles. The LB-agar plate counting method was usedto measure the number of conjugants and conjugative transfer frequency,and three replicate plates were set for each sterile conical bottleto ensure the reproducibility. Each treatment was conducted in ninebiological replicates and two repeated operations at least along withthe blank control without PA or the fractions.Horizontal ARG transfermediated by plasmid RP4 between bacteria strains in the presence ofPA and its distilled fractions was conducted using an optimized intragenericconjugative transfer model.46 Additionally, to confirm the successful transfer of plasmidsin donors to recipients, gel electrophoresis was applied to verifythe presence of plasmids in transconjugants. The detailed proceduresare provided in the Supporting Information (Text S1).In parallel,so as to eliminate the interference of bacterial spontaneous mutationand mutation induced by PA and its fractions, both the donor and recipientbacteria exposed to PA and its fractions were plated onto the transconjugant-selective plates.47 a LIVE/DEAD bacterial viabilityassay method,47 and a growth curve testingmethod.48 The 10%, 50%, and 90% MICs  were assessed bya broth microdilution method.47 The detailedprocedures are provided in the Supporting Information (Texts S2 and S3).In addition, to verify the effect of PA and itsthree distilledcomponents on the growth of donor, recipient, and transconjugant strains,three different assays were employed, that is, a LB-agar plate countingmethod,2.4To investigate the effectof PA acidity on ARG conjugation, three mating systems with desiredpH were set up, including the following: (1) the unadjusted-pH group:the treatments added with PA or its fractions (20 and 40 μL)without any pH adjustment; (2) the adjusted-pH group: the treatmentsadded with PA or its fractions, and the pHs were adjusted to 7.0 asthe control group by 0.1 M NaOH; (3) the no-PA group: the treatmentswithout PA or its fraction addition, and the pHs were adjusted asthe same as those containing corresponding PA or its fractions by0.1 M HCl. Besides pH adjustment, other operation procedures of theconjugation for these three treated groups were the same as thosein 2.544 In addition, 300 μL of glutathione , a scavenger of ROS, was addedinto the mating system to examine the role of intracellular ROS overproductioninduced by PA in the changed conjugation.44 The optimized concentration of GSH (300 μM), which was supersaturatedto reduce ROS production and cause little change in the donor andrecipient number, was selected according to a preliminary experiment. All experiments were measuredwith biological triplicates. The detailed procedures are providedin the Supporting Information (Text S4).To assess theeffect of oxidative stress induced by PA and its fractions on conjugation,the intracellular ROS level was measured by a 2′,7′-dichlorofluoresceindiacetate cellular ROS detection assay kit .2.636 Effects of PA and its fractions on cell membranepermeability were further investigated using propidium iodide  dye.44 In addition,300 μL of GSH (30 mM) was added in 30 mL of 10, 20, or 40 μLPA-exposed cell suspensions to verify the ROS effect induced by PAon the cell membrane permeability. All samples were conducted in biologicaltriplicates. The detailed procedures are provided in the Supporting Information (Text S5).The bacterialcell morphology in the presence of PA and its distilled componentswas characterized by transmission electron microscopy (TEM).38 The contents ofprotein and polysaccharide in the extracted EPS solution, two representativedominant components of EPS and playing important roles in cell surfacehydrophobicity and bacterial adhesion, were, respectively, analyzedusing a Detergent Compatible Bradford Protein Assay Kit  and phenol-sulfuric acid method.49 The details are described in the Supporting Information (Text S6).The heatextraction method was used to obtain EPS of bacteria as describedby previous research.E. coli cellsexposed with PA and the fractions were measured using a ZS90 zetasizer.14 Briefly, the bacterialsuspension treated with 20 and 40 μL of PA or its distilledcomponents in the mating system was washed with sterilized Milli-Qwater twice and the concentration was adjusted to 107 CFU/mLusing sterilized Milli-Q water. Finally, the zeta potentials of bacterialsuspension were measured.The zeta potentials of 2.7n ≥ 3). One-way analysis of variance (ANOVA) with Duncan’smultiple-comparison test (P < 0.05) and independent-samples t-test (P < 0.05) was adopted for significanceanalysis by the Statistical Product and Service Solutions software. Pearson correlation analysiswas performed by R software (version 4.0.3) with the “corrplot”package. Probit model was used to calculate the MIC10,MIC50, and MIC90 by SPSS 20.0.47 The bacterial growth curves were fitted using logisticgrowth model with Origin 2022.48All experimentaldata were expressed as mean ± standard deviation  in themating system significantly decreased the transconjugant number by74–85% (from 3.53 × 105 CFU/mL to 5.24 ×104–9.14 × 104 CFU/mL) comparedwith the control (Table S3). Inconsistently,PA significantly promoted the conjugative transfer frequency by 30–45%at 60–100 μL relative to the control . Thus,at a relatively high concentration of 60–100 μL, rangingbetween MIC50 and MIC90 (Table S1), the decreased abundance and activity of recipientand donor bacteria may substantially decrease the transconjugant numberdue to the antimicrobial effects  significantly increased the transconjugant number by 26–47%(from 3.53 × 105 CFU/mL to 4.43 × 104–5.19 × 104 CFU/mL) and the conjugative transferfrequency by 114–234% (from 6.79 × 10–5 to 1.45 × 10–4–2.27 × 10–4) (Table S3). Furthermore,the successful transfer of donor plasmids to recipients was verifiedby gel electrophoresis, showing clear bands of plasmids in the transconjugantswith the same size as those in the donor bacterial strains . In addition, no mutated bacteriawas observed on the transconjugant selection plates , further confirming that the resistanceacquisition of the E. coli colonieson the selective transconjugant plates was caused by the generationof transconjugants, rather than the spontaneous mutation of bacterialstrains exposed with PA. Taken together, these results proved ourhypothesis that PA amendment at the selected amount had a hermeticresponse  on intragenericconjugative transfer of ARGs.To test the effects of PA and its fractionson ARG conjugation, an LB-agar plate-based conjugation mating systemwas conducted. The results showed that PA at a relatively high amountof 40–100 μL , even though the donor and recipient bacterial concentrationswere decreased.38 These distinct amount-dependentpatterns of ARG conjugative transfer are mainly attributed to thedifferent chemical characteristics of these soil amendments, which could regulate ARG conjugation byinfluencing bacterial growth and population.39 Recently, a study evidenced that different types of dissolved biochars,the water-soluble components of biochars, at different amounts (1–100mg/L) posed significantly inhibited or promoted effects on conjugationof plasmid RP4.40 The effects were consistentwith that of PA, which is mainly explained by the complex compositionsof dissolved biochars such as humic acid-like substances, small moleculephenols, and organic acids. PAs, one of the by-products simultaneouslyderived from biomass pyrolysis for biochar production,29 containing similar chemical components with the dissolved biochars, would be responsible for thehormesis on ARG transfer, which will be discussed in the followingsection.Notably, the PA-promoted conjugative transfer alsotriggers thequestions regarding the potential risks of amendments playing non-negligible roles in spread of ARGs via HGT in soils orenvironments with similar characteristics ,particularly under the inappropriate amount. However, a study proposedthat low concentrations (1–5 mg/L) were the appropriate amountsfor CeO3.25 CFU/mL to 1.74 ×105 CFU/mL) at a relatively high amount (40 μL) butincreased the transconjugant number by 25–63% (from 4.64 ×105 CFU/mL to 5.82 × 105 CFU/mL in 10 μLand from 3.44 × 105 CFU/mL to 5.59 × 105 CFU/mL in 20 μL) at relatively low amounts (10 and 20 μL)(Table S4). Moreover, the hormesis effects ofPA and its fractions on the conjugation showed that PA had a strongerdegree of inhibition or promotion than its three fractions  with different relative contents . Thus, the promoted effect at arelatively low amount on the transfer showing the order of PA >F3≈ F2 > F1 and the inhibited effect at a relatively highamounton the transfer showing the order of PA > F3 ≈ F2 ≈F1 indicated the different roles of PA components in the conjugativetransfer.To distinguish the chemical componentsof pristine PA playing important roles in the conjugation, three distilledfractions of PA were first obtained at different temperatures of 98,130, and 220°C to examine their effects on the conjugative transfer.Similar to the pristine PA, these three distilled fractions also showedhormesis effects on the conjugative transfer 2. For exractions 2a,b. The25 were selected according to theirrelative contents . These fourrepresentative components also showed the hormesis effects on thetransfer of plasmid RP4  promoted the transconjugantnumber, respectively, by 39%, 49%, 12%, and 24%, following an orderof acetic acid ≈ 2-methoxy-phenolx > 2,6-dimethoxy phenol≈3-methyl-1,2-cyclopentanedione (Table S5). Contrarily,when the amounts of these four components increased to the inhibitorylevels ,the transconjugant number significantly decreased and followed anorder of acetic acid ≈ 2,6-dimethoxy phenol > 3-methyl-1,2-cyclopentanedione> 2-methoxy-phenolx (Tables S2 and S5). In previousstudies, these hormesis effects were also observed for antibiotics39 and phenolic compounds .51 However, freenitrous acid at the subinhibitory concentrations (<0.02 mg N/L)showed inhibited effects on plasmid RP4 conjugation within generaof E. coli K12 and E.coli HB101, caused by the limited adenosine triphosphateproduction and the down-regulated transfer genes via releasing intracellularFe2+ and decreasing intracellular Mg2+ levels.52 These results confirmed that the selected fourrepresentative components of PA variedly contributed to the hormesiseffects on the conjugation.To distinguish the roles of individual PA componentsin the conjugativetransfer, four representative components of PA, including acetic acid,two phenolic derivatives ,and 3-methyl-1,2-cyclopentanedione, determining PA functionality asa antimicrobial agent, antioxygen, a plant growth regulator, and afeed supplement,contents S1. Thesecontents S1. Thesecontents S1. Thesecontents S1. These47 determining bacterialactivity, oxidative stress, cell membrane permeability, and cellularmetabolism.52 The antibacterial activity of PA was stronger than that of its fractions, consistent withthe orders of hormesis effects induced by them on the conjugativetransfer  by damagingthe cell membrane, inhibiting energy metabolism, and denaturizingprotein.53 In the present study, the four representativecomponents effectively reduced the number of donor and recipient bacteriaat the inhibitory levels, showing the antibacterial activity orderof acetic acid > 2,6-dimethoxy phenol > 3-methyl-1,2-cyclopentanedione> 2-methoxy-phenolx . Additionally,the statistically significant correlations were observed between thecontents of these four representative components and the inactivationof donor or recipient strains , confirming that the bacteriostasis of these PA components at highamounts resulted in the inhibition of conjugation transfer. However,the contents of these four representative components showing an orderof F3 (85.6%) > F2 (68.2%) > F1 (67.4%) > PA (58.7%)  are not fully consistent with theorders of the inhibited conjugation. These results indicated thatthe potential synergistic effect of the individual components on theantimicrobial activity of PA partly played roles in regulating theconjugation, which should be examined in future. Consistently, thesub-MIC90 levels of acetic acid (0.001 mg/mL), 2-methoxy-phenolx(0.01–0.5 mg/mL), 2,6-dimethoxy phenol (0.1 mg/mL), and 3-methyl-1,2-cyclopentanedione(0.05–0.5 mg/mL) posed promotive effects on the conjugation in PA  or degradation products of fresh PA also exhibited antibacterial activity, probably contributingto the hormesis effects on the conjugation; this cannot be excludedin the present study, which needs to be further examined.53Besides the concentrationsor amount-dependenteffects, the effectsof these chemicals on conjugation are mainly shaped by their structure-dependentantibacterial activity,transfer 2.This s-phenolx S6. Addit3.356 The acidiccomponents of PA, such as acetic acid and 2-methoxy-phenol , endow PA and the distilled fractionswith the acidic characteristics (pH ≤ 2.81) , which significantly lowered the pH values (5.45–6.75)of the mating systems relative to the control (pH 7.0) (Table S6). Thus, it is reasonable to assume thatthe acidic pH of PA could play an important role in inhibiting conjugation.To test this hypothesis, three conjugation experiments with desiredpHs were conducted . Collectively, these results proved the hypothesisthat the decreased mating pH by acidic PA played a crucial role ininhibiting the conjugation of ARGs between E. coli, regardless of the high or low amount of PA.The process of ARG conjugative transferis highly sensitive to external environmental conditions .y-phenol S1b,c, eny-phenol S1b,c, eny-phenol S1b,c, eny-phenol S1b,c, eny-phenol S1b,c, en58 the key factors regulating ARGconjugation.57 Under the high amount of PA and its fraction except for F1, therecipient and donor numbers significantly decreased by 24–64and 31–56%, while their number partially restored after adjustingthe mating pH at 7.0, and decreased by 9–21 and 4–24%in the no-PA group compared to CK, respectively . Moreover, the statistically significant correlationswere observed between the mating pH and fold changes of recipientand donor numbers . These resultsevidenced that the decrease in matting pH by a high amount of PA causedstrong bacteriostasis. Extensive studies revealed that the E. coli strains in acidic conditions (pH 4.0 and5.5) generally resulted in low bacterial abundance and activity viadestroying the tertiary structure of the membrane protein by electrostaticeffects59 and suppressing DNA replicationby DNA depurination.60 The PA acidity isdetermined by its acidic components, such as acetic acid, 2-methoxy-phenol,2,6-dimethoxy phenol, and 3-methyl-1,2-cyclopentanedione, which alsohad antibacterial activity at inhibitory levels . For example, acetic acid reducedthe number of donors and recipients by 99% at 0.1 mg/mL, and 2-methoxy-phenolrespectively reduced by 75 and 83% at 1 mg/mL . These results further confirmed thatthe acidic components of PA such as acetic acid, 2-methoxy-phenol,2,6-dimethoxy phenol, and 3-methyl-1,2-cyclopentanedione under highlevels inhibited conjugation by their strong bacteriostasis. Consistently,a study demonstrated that acidification of manure from neutral (7.4)to acidic pH (4.8–5.4) promoted the degradation of sulfonamideantibiotics and decreased sul genes and sulfonamide-resistantbacteria .61 Thus, the risk of conjugative sul gene transfer was reduced, which was facilitated toreduce ARG risk in soils.61 On the contrary,another study found that the potential of gene transfer via geneticvectors  was promotedat acidic pH 4.0 and 5.0 but inhibited at alkaline pH 9.0 and 10.0,mainly attributed to the enhanced or inhibited propagation of tetracycline-resistantbacteria.57 These inconsistent effectsof acidic pH on the conjugation were mainly attributed to the differentpH sensitivities of the tested resistant strains (Pseudomonadaceae vs Acetobacter) in the two studies.However, in the present study, the E. coli strains showed poor adaptability to acidic pH via the decreasedbacterial activity in low pH (5.45–6.45) caused by PA, whichfirst provided the direct evidence that the acidic characteristicof PA amendment can inhibited the conjugative transfer of ARGs viaits bacteriostasis.Particularly, pH is generallyclosely related tobacterial abundanceand activity,p-aminophenol,and phenol) at sub-inhibitory levels could promote ARG conjugationby the increased ROS production, membrane permeability, and intercellularcontact but inhibited the conjugation via bacteriostasis above theMIC levels.44 Therefore, the promoted conjugationby PA may be regulated by (1) intracellular ROS production,51 (2) membrane permeability,44 and (3) intercellular contact,38 but the inhibited conjugation by PA may be regulated by (1) theinhibited recipient and donor growth, (2) destroyed antioxidant system,(3) damaged cell membrane, and (4) broken pilus, which are furtherverified in the following sections.Notably, although the pHs of the adjusted-pHgroup were the sameas the CK groups, the transconjugant number in the adjusted-pH groupunder a high amount of PA was still lower compared to CK 4. Furthe3.4•O2–, •OH, and H2O2, could stimulate oxidative stress,break biomolecules , activateSOS response, and even cause cell death, thus regulating the ARG conjugation.51 In this study, the amount-dependent increase in intracellular ROSlevels was found in the E. coli strainsexposed with PA and the fractions, and the increasing degree of ROSvia PA was stronger than its three fractions . ROS may directly break double-strandDNA or indirectly damage DNA by oxidizing the deoxynucleotide pool,inducing SOS response and repair mechanisms to enhance the successfulrecombination of exogenous genes,62 thusstimulating the conjugation process.63 These resultsconfirmed that the low amount of PA and the fractions promoted theconjugation via the ROS overproduction. The overproduction of intracellularROS could induce oxidative stress, thus disrupting the intracellularredox balance.62 Notably, although PA andthe fractions induced the higher ROS levels at a high amount of 40μL than the low amounts of 10 and 20 μL . To further determine whether the observed increasein ROS production was related to the intrageneric conjugative transfer,the effect of GSH, a widely used ROS scavenger,64 on plasmid RP4 conjugation was examined , afteradding GSH, the conjugative transfer frequency and the number of recipientand donor cells also had no significant difference with those in thecontrol without PA group (S9), further confirmingthat a high amount of PA and its fractions resulted in cell deathby exceeding ROS levels to inhibit the conjugation.ROSin bacterial cells induced by external environment stress, including ractions 5a, in liractions 2a,b. Impractions S7. ROS mnd 20 μL 1a and 2aractions S7. ROS mractions S7. ROS mPA group 5b and S9Figure S7). The fold changes of ROS levels positivelycorrelated with the contents of acetic acid, 2-methoxy-phenolx, 2,6-dimethoxyphenol, and 3-methyl-1,2-cyclopentanedione of PA , implying that these four representative chemicalcomponents play important roles in the ROS overproduction. Extensivestudies showed that phenolic compounds such as thymol, eugenol, andcarvacrol could destroy the antioxidant defense systems of bacterialcells by decreasing antioxidant enzyme activity  and causing excessive ROS accumulation.65 These results displayed that the representative chemical components,particularly phenolic compounds such as 2-methoxy-phenolx and 2,6-dimethoxyphenol detected in the PA, played important roles in the ROS overproduction.Furthermore, the acidic conditions could also enhance the ROS accumulationby inhibiting antioxidant enzyme activity  and destroyingelectron transport chains in bacterial cells, resulting in leakageof electrons to oxygen prematurely and ROS formation.67 Pearson correlation coefficient analysis showed the negative correlationbetween the fold changes of ROS levels and the pH values , implying that the decreased pH causedby acidic PA also induced the ROS overproduction in E. coli cells. Together, these results validatedthat the moderate ROS level of E. coli induced by PA at relatively low amounts promoted the RP4 plasmid-mediatedconjugative transfer within E. coli genera, while the exceeding level of ROS induced by high amountsof PA resulted in cell death to inhibit the conjugation, and the ROSoverproduction was mainly due to the representative components ofPA  and the decreased pH.To further investigate the potential relationshipbetween ROS overproductionand PA components, Pearson correlation analysis was conducted (3.531 The cell membrane structure of the mixed donorand recipient strains exposed to PA and its fractions was first assessedby TEM (S10). In the untreated group, the E. coli strains showed a normal cellular structurewith intact cell membranes, a smooth surface, and compact cytoplasm,and the cells were dispersed with limited physical contact (S10). At the low amounts (10 and 20 μL)of PA, the cells were obviously observed with damaged cell membranes,emerged pores, unclear cell boundaries, and enhanced cell-to-cellcontact (S10a), implying that the bacterialmembrane permeability enhanced, which may favorable to ARG conjugation.51 Furthermore, the bacterial membrane permeabilitywas further quantified using flow cytometry (E. coli strains exposed with low amounts (10 and 20 μL) of PA andthe fractions, and PA had a stronger degree of enhancement on themembrane permeability than its three fractions . These results demonstrated that the low amount of PA and fractionspromoted the conjugation via the enhanced cell membrane permeability(S10). Consistently,this was also why more severely damaged cell membrane and shrunkenor leaky cytoplasm were found in the TEM images (S10), supported by the LCSM images of the donor and recipient and bacteria growth curvesof the donor, recipient, and transconjugant . Moreover, bacterial pilus generally takescharge of competence for DNA uptake, motility, and cell surface adhesions,68 which may facilitate the resistance plasmidtransfer between bacteria. The complete pilus was found on the surfaceof untreated bacterial cells in the control group, while obvious pilusfractures were observed for the bacterial cell treated with the highamount of PA (S10), thus impeding the successfultransfer of ARGs. These results demonstrated that a moderate increasein cell membrane permeability induced by a low amount of PA facilitatedthe conjugative ARG transfer, while the severe cell membrane damage,pilus fracture, cytoplasmic shrinkage, and leakage caused by a highamount of PA resulted in cell death to inhibit conjugation.Bacterialcell membrane is an important barrier to prevent extracellularchemical materials and genes from free accessing cells.ytometry 5d. Speciractions 5d, consiractions 2a,b. Fureability1a and 2aractions S7. Theseractions S7. These62 To furtherverify the increased bacterial membrane permeability induced by ROSoverproduction under PA exposure, the membrane permeability of bacterialcells added with GSH was examined. Regardless of the low or high amountof PA exposure, the cell membrane permeability in the GSH treatmentswas significantly lower relative to those without GSH addition andshowed no significant difference compared with those in the controltreatment without PA addition . Additionally, significant positive correlations were observedbetween the fold changes of ROS and cell membrane permeability . These results illustrated that theincreased ROS levels induced by PA exposure enhanced the membranepermeability of the E. coli strains.Previous studies have shown that sub-inhibitory concentrations (0.04–1.00mg/L) of chloroxylenol, as an aromatic organic compound containingphenol, could increase cell membrane permeability by the membranelipid peroxidation to stimulate the plasmid RP4 transfer between E. coli DH5α and Pseudomonas HLS-6.51 Toxic chemicals, particularlyhydrophobic molecules ,could accumulate within the membrane lipids, participate in chemicalinteractions between the fatty acyl chains, and damage the membranephospholipids to facilitate cell membrane permeability.69 Furthermore, positive correlations were foundbetween the fold changes of cell membrane permeability and the contentsof acetic acid, 2-methoxy-phenolx, 2,6-dimethoxy phenol, and 3-methyl-1,2-cyclopentanedionein PA . These results displayedthat these four representative components detected in the pristinePA contributed to the increased cell membrane permeability. Additionally,the acidic conditions also could enhance the cell membrane permeabilityby increasing the proportion of unsaturated fatty acids, particularlyC18-1w9c and C19-Cyc, which were the key metabolites in the synthesisof fatty acids and the substrates for the synthesis of unsaturatedfatty acids.70 The negative correlationwas observed between the fold changes of cell membrane permeabilityand the pH levels , suggestingthat decreased pH by acidic PA also contributed to increase the E. coli cell membrane permeability. In sum, the increasedROS levels induced by these critical components and low acid pH inPA and its fractions enhanced membrane permeability, thereby promotingconjugative transfer at low amounts.ROS could react with polyunsaturated fatty acids located on bacterialcell membrane, leading to excessive oxidation of lipids, thereby enhancingthe membrane permeability to facilitate the plasmid transfer fromdonor to recipient bacteria.3.649 EPS, majorly composed of proteins, polysaccharides, extracellularDNA, and lipids,5 plays important rolesin biofilm formation and bacterial gene transfer by promoting intercellularcontact.49 TEM images showed that PA exposure stimulatedthe cell-to-cell contact with suspicious surrounding secretions (S10). Furthermore, EPS content of the mixed E. coli strains was measured (S12a–c). PA exposure at 20 μL significantly increased the contentsof proteins and polysaccharides, respectively, by 40 and 13% , and the total EPS contents increasedby 29% . The ratio of proteinsto polysaccharides (PN/PS) in EPS, reflecting cell surface hydrophobicitydue to the strong hydrophilicity of polysaccharides with rich hydrophilicgroups  and hydroxyl (−OH) groups),49 also increased in the PA treatments , the conjugation was still inhibited . Additionally,bacteria under the exogenous stress, such as low pH or chemicals,could also synthesize and secrete EPS by upregulating the expressionof EPS production-related genes,72 which act as a barrierto reduce the destruction of the external adverse environment on bacteriathemselves.49 Pearson correlation coefficientanalysis showed the significant correlation between the fold changesof EPS or the PN/PS values and the content of 2,6-dimethoxy phenoland 3-methyl-1,2-cyclopentanedione or the pH values . These results suggested that the chemical componentsof PA, especially 2,6-dimethoxy phenol and 3-methyl-1,2-cyclopentanedione,and the acidic pH by PA contributed to synthesizing and secretingEPS of E. coli.Direct active intercellular contact is the prerequisitefor successful conjugative ARG transfer. and 13% S12a,b, a and 13% S12a,b, a and 13% S12a,b, a and 13% S12a,b, a73 Hence, the cell surface zeta potential was further measured (S12d). Compared with the control, the surfacenegative charge of E. coli cell exposedwith PA at 20 μL significantly decreased (S12d). Similarly,the three fractions also posed decreased effects on the surface negativecharge, but the decreased levels were lower than those in the PA treatments(S12d), consistent with the promoted order onthe conjugative transfer  of PA also decreased thecell surface charge (S12d), although massive recipientand donor bacteria were inactivated and the conjugation was inhibited. A previous study showed that soil acidification (pH 5) decreasedthe surface negative charge of B. subtilis, a type of soil-dwelling bacteria beneficial to plant and soil animalgrowth, by the protonation of its membrane proteins.66 These findings suggested that the acidic components ofPA decreased the E. coli surface negativecharge via the protonation of cell membrane proteins, thus promotingthe intercellular contact by decreasing intercellular electrostaticrepulsion to facilitate the ARG conjugation. Furthermore, soil biofilms,a supracellular structures formed by microorganisms encasing in theself-produced matrix of EPS, represent the predominant microbial lifestylein soils.5 Soil biofilms have a high bacterialdensity to allow intercellular contact, which are deemed the hotspotsof ARG spread and HGT occurrence.75 However, whetherthe PA could promote biofilm formation to enhance the ARG conjugativetransfer is unclear, which remains to be explored in the future. Furthermore,the changes of microbial communities in the soil environment couldalso significantly affect the process of HGT in PA-applied soils.29 The effect of soil microbial communities onARG spreading via HGT after applying PA amendments could be furtherexplored with the help of microfluidics and fluorescence in situ detectiontechnology in the future.The outer cell membrane is directly interacted with the surroundingenvironment. Thus, cell surface characteristics  are easily affected by the surroundingconditions such as nanoparticles,4E. coli. The inhibited conjugative plasmid RP4 transferat high amounts (40–100 μL) of PA is ascribed to on conjugative transfer of plasmid RP4 within the genus ribed to 6 (1) theribed to 6 (1) theThese results provide new insights on the effectsof PA amendmenton ARG conjugation and convincing evidence for alleviating the spreadof soil ARGs using PA amendments at the appropriate application amountto be a practical strategy. Considering the hormesis of PA on theconjugation, our results highlighted the necessity to optimize theamount of soil amendments to alleviate ARG pollution in soils by combatinghorizontal transfer of ARGs. Moreover, the PA-promoted conjugationalso triggered questions regarding the potential risks of PA amendmentsand other soil amendments having antibacterial activity  applied at inappropriate levels in the spreadof ARGs via HGT in soil ecosystems, which should be carefully considered.Considering that the representative components of PA  at non-lethal levels played critical roles inenhanced conjugation, more studies are warranted on optimizing thebacteriostasis of PA by selecting the appropriate feedstock and distilledtechnology to enhance the efficiency of PA amendments in remediatingsoil ARG pollution. Furthermore, based on PA-promoted conjugationby enhancing ROS production, membrane permeability, and EPS production,the co-application of PA with some antioxidants and EPS scavengers  could be feasible strategies to lower the potential risksof PA in facilitating ARG spread through conjugation. However, onlyone woody waste-derived PA was selected in the present study to investigateits effect on ARG conjugation among intrageneric bacteria using apure culture model; further studies are warranted to examine the effectsof more types of PA amendments on intrageneric and intergeneric conjugativetransfer or other HGT routines among the microbial communities in practical soil ecosystems.


Citrus sinensis seedlings were supplied with a nutrient solution containing 15 (control) or 0 (nitrogen (N) deficiency) mM N for 10 weeks. Extensive metabolic and gene reprogramming occurred in 0 mM N-treated roots (RN0) to cope with N deficiency, including: (a) enhancing the ability to keep phosphate homeostasis by elevating the abundances of metabolites containing phosphorus and the compartmentation of phosphate in plastids, and/or downregulating low-phosphate-inducible genes; (b) improving the ability to keep N homeostasis by lowering the levels of metabolites containing N but not phosphorus, upregulating N compound degradation, the root/shoot ratio, and the expression of genes involved in N uptake, and resulting in transitions from N-rich alkaloids to carbon (C)-rich phenylpropanoids and phenolic compounds  and from N-rich amino acids to C-rich carbohydrates and organic acids; (c) upregulating the ability to maintain energy homeostasis by increasing energy production  and decreasing energy utilization for amino acid and protein biosynthesis and new root building; (d) elevating the transmembrane transport of metabolites, thus enhancing the remobilization and recycling of useful compounds; and (e) activating protein processing in the endoplasmic reticulum. RN0 had a higher ability to detoxify reactive oxygen species and aldehydes, thus protecting RN0 against oxidative injury and delaying root senescence. Citrus grandis) orchards were sub-optimum in hydrolyzable N and deficient in foliar N, respectively.Plants need nitrogen (N) to synthesize nucleic acids (NAs), amino acids (AAs), proteins, and other N-containing compounds needed to maintain their life cycle. N fertilizers play a vital role in citrus growth and development ,2. A posIn higher plants, N deficiency has remarkable impacts on a series of physiological and biological processes, including growth, nutrient and water uptake, photosynthesis, pigment biosynthesis, carbon (C) and N metabolism, C and N partitioning between roots and shoots, energy metabolism, lipid metabolism, reactive oxygen species (ROS) formation and removal, NA metabolism, phytohormone biosynthesis, cell wall metabolism, senescence, and secondary metabolism ,11,12,13To deal with N starvation, plants have developed a series of adaptive strategies, including increasing C and N fractions in roots and the root-to-shoot dry weight (DW) ratio (R/S); lessening growth; altering root architecture; improving N uptake, N use efficiency (NUE), and/or remobilization efficiency (NRE); making a transition from N-rich to C-rich compounds; and delaying root senescence ,10,13,14Panicum miliaceum Panicum mdigotica . Few resdigotica , soybeandigotica , poplar digotica , barely digotica , maize  , and L-glutamine in RN0 implied that N deprivation impaired root N assimilation. This was consistent with the results that N deprivation led to decreased activities of five enzymes (including NR) related to N assimilation, and levels of N  was induced in A. thaliana roots from day 2 after N deprivation, and that NRT2.5 deficiency caused a reduced uptake of nitrate in the high-affinity range. Further analysis demonstrated that NRT2.5 was necessary to support plant growth under N limitation via guaranteeing the efficient uptake of nitrate together with NRT2.1, NRT2.2, and NRT2.4, and via participating in nitrate loading into the phloem during its remobilization. Hsu and Tsay  2 (NIA2) and NRT1.1 and reducing H2O2 accumulation due to increased catalase (CAT) activity. In addition, we isolated eight downregulated and seventeen upregulated genes involved in cellular N compound catabolic process (GO:0044270) in RN0, implying that N deprivation improved N compound degradation. This agreed with our findings that more downregulated than upregulated N-containing compounds were detected in RN0 (NRT2.4 (Cs8g16010 and orange1.1t02415) and NRT2.5 (Cs7g09040) related to nitrate absorption and decreased the expression of root NRT1.1 involved in nitrate transport from roots to shoots, thus elevating N fraction in roots [CLC-a (Cs2g25150) in RN0, thus facilitating the maintenance of nitrate homeostasis. N deficiency improved NUE, as indicated by upregulated proteinNLP7 (Cs5g01890) in RN0 and NRE, as indicated by the increased degradation of N compounds.In addition to elevating R/S, plants have developed diverse mechanisms to maintain N homeostasis ,10. Lezhand Tsay  indicaten et al.  observedg et al.  reportedn et al.  indicaten et al.  found thn et al.  reportedi et al.  reportedu et al.  observedg et al.  demonstrd in RN0 . OverallN deficiency can cause leaf senescence , but dateukaryotic initiation factor 4A-9 ), and other DEGs related to protein biosynthesis  in RN0 , and five ribosomal protein genes , and reduced abundances of substrates (AAs) for protein biosynthesis.We identified ten downregulated and fourteen upregulated genes related to protein folding , ribosome biogenesis , translation initiation factor activity  in RN0 , as well) in RN0  in RN0. We obtained eighteen (fifteen) downregulated and twenty (ten) upregulated genes (metabolites) involved in biosynthesis of AAs (seventeen (fourteen) downregulated and eighteen (nine) upregulated genes (metabolites)), arginine biosynthesis  downregulated and two (two) upregulated genes (metabolites)), proline biosynthetic process , lysine biosynthesis  downregulated and one (four) upregulated gene (metabolites)), valine, leucine and isoleucine biosynthesis  downregulated and one (zero) upregulated gene (metabolite)), and phenylalanine, tyrosine and tryptophan biosynthesis  downregulated and three (two) upregulated genes (metabolites)) in RN0, as well as seven (three) downregulated and eight (four) upregulated genes (metabolites) involved in cellular AA catabolic process , valine, leucine and isoleucine degradation , aromatic amino acid (AAA) family catabolic process , and lysine degradation  downregulated and one (four) upregulated gene (metabolites)) in RN0 . AdditioAs a sink organ, roots need carbohydrates provided by leaves to grow and develop. Because N starvation caused a reduction in root growth and a rise in root C fraction ,8, C, caC. sinensis seedlings [phosphoenolpyruvate carboxykinase , triosephosphate isomerase, cytosolic , and β-fructofuranosidase, insoluble isoenzymeCWINV1 (Cs4g18340), and/or increased utilization for glycolysis (energy production) and starch biosynthesis. In addition, we identified three upregulated genes (granule-bound starch synthase 1 (orange1.1t00566) and two glucose-1-Pi adenylyltransferase (Cs2g18800 and Cs8g07230) genes) in starch biosynthesis and three downregulated sucrose synthase genes  in RN0, implying that N deficiency increased and decreased starch and sucrose biosynthesis, respectively, in roots. This agreed with the report that N deprivation led to increased starch accumulation but decreased glucose, fructose, and sucrose accumulation in C. sinensis roots [N-deficient plants enhance their ability to acquire N by improving carbohydrate allocation to support root growth and optimizing root morphology . More upeedlings . N deficNV1 Cs4g1340, and/NADP-dependent malic enzyme  and phosphoenolpyruvate carboxylase 2 ) and twenty-four upregulated ) genes, and one downregulated and three upregulated metabolites associated with glycolysis/gluconeogenesis, pyruvate metabolism, and the TCA cycle in RN0, which implied that OA biosynthesis might be enhanced in RN0. This disagreed with our findings that N deprivation decreased the activities of eight acid-metabolizing enzymes  in roots [We isolated fifteen downregulated (including in roots  and thatin roots .triacylglycerol lipase SDP1 (orange1.1t00747)) related to linoleic acid metabolism (ko00591) in RN0. LOX catalyzes the oxygenation of polyunsaturated FAs with the generation of hydroperoxides. In plants, elevated LOX activity has been shown to be associated with increased MDA, a secondary end product of polyunsaturated FA oxidation [DkLOX3 in A. thaliana promoted dark-induced senescence and increased EL and MDA accumulation in leaves [Plant cells can respond to various stresses by changing the composition of plastic membrane lipids and plastic outer membrane lipids by lipid remodeling and maintaining the fluidity, stability, and integrity of membranes . We screxidation . The oven leaves . Taken tn leaves  and lowen leaves .C. sinensis seedlings relative to 15 mM treatment [OsPHT1;3 led to elevated levels of P in rice shoots and roots, and that OsPHT1;3 played a crucial role in the uptake and root-to-shoot translocation of Pi [probable inorganic Pi transporter 1-3 , inorganic Pi transporter 1-11 , and probable inorganic Pi transporter 1-7 ) in RN0, implying that N deficiency improved Pi uptake. This agreed with increased P uptake per root DW and P levels in leaves, stems, and roots of N-deficient C. sinensis seedlings [probable ubiquitin-conjugating enzyme E2 24 , which inhibited Pi uptake via facilitating the degradation of Pi transporter PHO1 and PHT1 proteins [TSub_g9430.t1, one gene similar to PHT4;1 and PHT4;4, was one of the most upregulated genes in Trifolium subterraneum roots after Pi addition, and that the subcellular accumulation of Pi in globular structures (possibly including plastids) might contribute to cytosolic Pi homeostasis under high P. Glycerophosphodiester phosphodiesterase (GDPD) participates in the release of inorganic Pi from PLs during Pi limitation [2+/H+ transporters CAX3 and CAX1 contributed to Pi homeostasis by inhibiting a subset of low-Pi-responsive genes (LPRGs) in A. thaliana roots and shoots [GDPD3 (Cs7g02950) and upregulation of PHT4;4 (orange1.1t00376), PHT4;1 (Cs8g19980), and CAX3 (Cs8g20150 and Cs6g08320) in roots, suggested that N deficiency increased Pi compartmentation in plastids and/or Golgi apparati, inhibited LPRG expression, and decreased the release of inorganic Pi from PLs, thereby contributing to Pi homeostasis. Additionally, N deprivation lowered the expression of many low-Pi-inducible genes (LPIGs), including histidine kinase 4 (Cs2g17860), phosphoenolpyruvate carboxykinase (ATP) (Cs1g20920), protein DETOXIFICATION 43 , SPX domain-containing proteins (Cs4g17870 and orange1.1t00194), multicopper oxidase LPR1 (orange1.1t04786 and Cs1g14690), protein TRANSPORT INHIBITOR RESPONSE 1 , protein PHR1-LIKE 2 (orange1.1t03293), and acid phosphatases  in roots , sixteen downregulated and twenty-five upregulated genes in nucleotide biosynthetic process (GO:0009165), three downregulated and one upregulated gene in nucleotide salvage (GO:0043173), and five downregulated and twelve upregulated genes in nucleotide catabolic process (GO:0009166) in RN0 , as wellIn summary, root primary metabolism exhibited obvious adaptive responses to N deficiency, including: (a) delaying root senescence; (b) elevating the ability to keep Pi homeostasis by increasing the abundances of P-containing metabolites, Pi compartmentation in plastids, and/or lowering the expression levels of LPIGs; (c) upregulating the ability to keep N homeostasis by decreasing the abundances of metabolites containing N but not P, enhancing the degradation of N compounds, R/S, and the expression of genes associated with N uptake, and leading to a transition from N-rich AAs to C-rich carbohydrates and OAs; and (d) improving the ability to maintain energy homeostasis by increasing energy production and decreasing energy utilization.In plants, N availability has a huge impact on the biosynthesis of SMs ,64. We iMoringa oleifera callus tissues. Taken together, N deficiency lowered the levels of L-phenylalanine, L-tyramine, L-lysine, L-glutamine, and L-ornithine, thus reducing biosynthesis and the accumulation of alkaloids. However, the levels of all three differentially abundant plumerane  in roots increased under 0 mM N  and lignin biosynthetic process GO:0009809) in roots . Obvious and lign09 in rooTo conclude, N deprivation led to a transition from N-rich alkaloids to non-N-containing (C-rich) phenylpropanoids and phenolic compounds  in roots.A. thaliana [peroxidases, seven glutathione S-transferases (GSTs), L-ascorbate peroxidase (APX) 2 , annexin D1 , four thioredoxins , three glutaredoxins , six L-ascorbate oxidases (AOs), monodehydroascorbate reductase , and GDP-mannose 3,5-epimerase 1 ) and fifty-nine upregulated  genes (Cs1g20630 and Cs1g20650), type IV inositol polyphosphate 5-phosphatase 9 , CAT isozyme 1 , glyoxylate/succinic semialdehyde reductase(GLYR) 1 , peptide methionine sulfoxide reductase (MSR) B5 , nudix hydrolase(NUDX) 2 , two TRXs (novel.84 and Cs6g04000), nucleoredoxinx , and two glutathione hydrolase 3 (GGT3) genes (Cs4g13090 and Cs4g13100), GDP-L-galactose phosphorylase (GGP) 1 ) associated with ROS metabolic process , antioxidant activity , response to oxidative stress , cell redox homeostasis , ascorbate (ASC) and aldarate metabolism , and glutathione metabolism (ten downregulated and sixteen upregulated genes) in RN0, as well as five upregulated metabolites associated with ASC and aldarate metabolism and five downregulated and one upregulated metabolite related to glutathione metabolism in RN0 , one downregulated and five upregulated genes related to aldehyde biosynthetic process (GO:0046184) , and elethaliana . Therefom in RN0 . As shown plants . Golani n plants  observed2O2 removal and lignin polymerization [peroxidases agreed with the reduced accumulation of lignin in RN0  had little impact on the whole leaf ASC level and huge impact on apoplastic ASC level. Reduced AO activity caused an increase in the ratio of ASC to dehydrateascorbate (DHA), while increased AO activity led to the oxidation of the apoplastic ASC pool. There was a positive relationship between AO activity and plant height (biomass). Yamamoto et al. [AO under salt-stress conditions led to a relatively low accumulation of H2O2 and a high ratio of symplastic and apoplastic ASC/DHA, and antisense AO tobacco and A. thaliana T-DNA mutants had enhanced tolerance to salt, H2O2, and methyl viologen (MV), and reduced APX activity. The downregulation of six AOs in roots induced by N deficiency might inhibit root growth, but it could be an adaptive strategy to N deficiency by protecting roots from oxidative damage. The downregulation of six AOs also agreed with the reduced expression of APX2 in RN0. Wang et al. [GGP displayed elevated tolerance to MV-mediated oxidative stress and ASC accumulation. The overexpression of an alfalfa GME involved in ASC biosynthesis conferred A. thaliana tolerance to acid, drought, and salt by preventing oxidative damage due to increased ASC accumulation [In plants, enzymes  and metabolites (GSH and ASC) related to glutathione metabolism, and ASC and aldarate metabolism play a crucial role in stress tolerance . GSTs clubstrate . Transgee stress ,85. GGTse stress . The mori et al.  reportedo et al.  indicateg et al.  reportedmulation .2)) and three upregulated  nicotinate) vitamins in RN0  downregulated and thirteen (six) upregulated genes (metabolites) associated with nicotinate and nicotinamide metabolism (ko00760), biotin metabolism (ko00780), and riboflavin metabolism (ko00740) in RN0, as well as one downregulated , aldo-keto reductases (AKRs), and alkenal reductases, GSTs also function in the detoxification of aldehydes by catalyzing the conjugation of aldehydes to GSH . We obtaTo conclude, N deprivation had a huge influence on ROS and aldehyde formation and removal in RN0, but RN0 could keep a higher ability to detoxify ROS and aldehydes, thus protecting RN0 against oxidative injury, as shown by reduced MDA accumulation and EL.ABC transporters can mediate the transport of mineral ions, phytohormones, SMs, sugars, lipids, peptides, OAs, phytate, defense-related chemicals, and toxic compounds, thereby playing a vital role in organ growth, plant nutrition and development, response to abiotic stresses, defense, detoxification, and plant–environment interaction ,93. We oWe detected fewer total, downregulated, and upregulated DAMs in RN0 than 0 mM N-treated leaves (LN0), but more total, downregulated, and upregulated DEGs in RN0 than LN0. There were 143, 54, and 39 common total, downregulated, and upregulated DAMs between RN0 and LN0, respectively, as well as 542, 243, and 132 common total, downregulated, and upregulated DEGs between both, respectively. Among the common total DAMs and DEGs, 50 DAMs and 167 DEGs displayed the opposite trends. Enriched GO terms in BP, MF, and CC, and enriched KEGG pathways for DEGs and DAMs were greater in RN0 than LN0 . AdditioOver 93% (79%) of the enriched KEGG pathways (GO terms) obtained in LN0 were identified in RN0 . ObviousC. sinensis (L.) Osbeck) seedlings were used in this study because C. sinensis is polyembryonic seed development. Many embryos are developed directly from the maternal nucellar tissue of the ovule surrounding the sexual embryo sac and have the same genetic constitution as the female plant [C. sinensis seedlings were slightly less tolerant to N deficiency than C. grandis seedlings, and N deficiency induced an increase in R/S, which was greater in the former than the latter [2SO4, 2.5 mM CaCl2, 2 mM MgSO4, 1 mM KH2PO4, 2.5 mM KNO3, 2.5 mM Ca(NO3)2, 1.25 mM (NH4)2SO4, and 5 mM NH4Cl) and micronutrients 6Mo7O24). Six weeks after germination, uniform seedlings were transplanted into 6 L pots (two plants per pot−1) containing sand. Seedlings were grown in a glass greenhouse under natural conditions at Fujian Agriculture and Forestry University, Fuzhou, China , with annual average sunshine hours, relative humidity, and temperatures of ~1600 h, 76%, and 20 °C, respectively. One week after transplanting, each pot was fertilized thrice weekly with quarter-, half-, and full-strength nutrient solutions for one, two, and three weeks, respectively, until part of the nutrient solution started to leak out of the hole at the bottom of the pot (500 mL pot−1). Seven weeks after transplanting, each pot was irrigated thrice weekly with a nutrient solution containing 15 (control) or 0 (N deficiency) mM N  for 10 weeks [2. Frozen samples were stored at −80 °C until the extraction of RNA and metabolites. The roots of seedlings without sampling were used to measure N, SAPR, EL, MDA, total phenolics, and lignin.‘Xuegan’  were used for the N assay. N was measured by indophenol blue spectrophotometry . MDA was determined by referring to Hodges et al. . SAPR wawww.shimadzu.com.cn/, accessed on 15 January 2022; MS, Applied Biosystems 4500 Q TRAP, www.appliedbiosystems.com.cn/, accessed on 15 January 2022). Each treatment contained three biological replicates. A metabolite with both a variable importance in projection (VIP) > 1 in the OPLS-DA and a |log2(fold change)| > 1 was considered differentially abundant. DAMs were annotated using the MetWare metabolite database and KEGG compound databases [Widely targeted metabolome was made by the Wuhan MetWare Biotechnology Co., Ltd.  with an ultra-performance liquid chromatography electrospray ionization–tandem mass spectrometry (UPLC-ESI-MS/MS) system  and actin (Cs1g05000) as the internal standards, qRT-PCR was performed in two technical replicates and three biological replicates [Twenty-seven DEGs were randomly selected for qRT-PCR confirmation. 2 values.Integrated analysis was carried out according to Peng et al.  after cot-test between two means was performed by SigmaPlot 10.0  at p < 0.05. PCA, HCA, and OPLS-DA were made using R software .An unpaired NRT2.4, NRT2.5, NRT1.1, NLP7, and LOX and metabolic pathways such as PPER, arginine and proline metabolism, and ABC transporters might play a role in root N deficiency tolerance. Additionally, we observed some differences and similarities in DEGs and DAMs between roots and leaves. Thus, this study provided novel evidence on the adaptive mechanisms of plant roots to prolonged N deficiency and provided a theoretical basis for guiding N management in citrus.Our findings clearly indicated that RN0 had extensive metabolic and gene reprogramming to deal with N deficiency , includi


Non-alcoholic steatohepatitis (NASH), an advanced subtype of non-alcoholic fatty liver disease (NAFLD), has becoming the most important aetiology for end-stage liver disease, such as cirrhosis and hepatocellular carcinoma. This study were designed to explore novel genes associated with NASH.Here, five independent Gene Expression Omnibus (GEO) datasets were combined into a single cohort and analyzed using network biology approaches.11 modules identified by weighted gene co-expression network analysis (WGCNA) showed significant association with the status of NASH. Further characterization of four gene modules of interest demonstrated that molecular pathology of NASH involves the upregulation of hub genes related to immune response, cholesterol and lipid metabolic process, extracellular matrix organization, and the downregulation of hub genes related to cellular amino acid catabolic, respectively. After DEGs enrichment analysis and module preservation analysis, the Turquoise module associated with immune response displayed a remarkably correlation with NASH status. Hub genes with high degree of connectivity in the module, including CD53, LCP1, LAPTM5, NCKAP1L, C3AR1, PLEK, FCER1G, HLA-DRA and SRGN were further verified in clinical samples and mouse model of NASH. Moreover, single-cell RNA-seq analysis showed that those key genes were expressed by distinct immune cells such as microphages, natural killer, dendritic, T and B cells. Finally, the potential transcription factors of Turquoise module were characterized, including NFKB1, STAT3, RFX5, ILF3, ELF1, SPI1, ETS1 and CEBPA, the expression of which increased with NASH progression.In conclusion, our integrative analysis will contribute to the understanding of NASH and may enable the development of potential biomarkers for NASH therapy. Non-alcoholic fatty liver disease (NAFLD) is likely to become the most common chronic liver disease, affecting about 25% in the adult population . It is cPNPLA3, TM6SF2, GCKR, MTARC1 and HSD17B13) that promote NAFLD risks in humans  have revealed genetic variants in several loci  associated with NASH by integrating transcriptomic data with biological network analysis between normal liver tissues and NASH tissues. We obtained five liver transcriptome datasets from the Gene Expression Omnibus (GEO) database . We firshttp://www.ncbi.nlm.nih.gov/)  database (ih.gov/) . We searih.gov/)  platformp less than 0.05, and the absolute difference of means more than 0.3. Gene ontology (GO) and Reactome enrichment analyses were performed for DEGs using hypergeometric test, which is conducted by the python package gseapy , all gene sets of GO term and Reactome pathway were obtained from database source of Enrichr  method with oligo/Bioconductor . For RNA Enrichr . Only GOn = 164) based on the median absolute deviation in the MergeCohort were kept for WGCNA and tested by the WGCNA R package (in) was defined to measure for each gene on the base of its correlation with the remaining genes in a given module. Genes with highest Kin are identified as hub genes. Cytoscape version 3.8.2 was used for visualization. In order to understand the extent of module preservation in MergeCohort, a publicly available expression profiling of high throughput RNA sequencing dataset GSE135251 including 10 controls, 51 NAFL and 155 NASH was used, processed as described above. Module preservation analysis was carried out by using Module preservation function in WGCNA package introduced by Langfelder et al.  and we presented top 20 for each disease-associated module. Additionally, to obtain regulatory information of transcription factors (TFs) and target genes, Transcriptional Regulatory Relationships Unraveled by Sentence based Text mining (TRRUST) v2 database (https://www.grnpedia.org/trrust/) (https://github.com/zqfang/gseapy). In addition, ChIP-X Enrichment Analysis 3 (ChEA3) database (https://maayanlab.cloud/chea3/)   were suptrrust/) , conduct/chea3/)  was adopn = 2,000) were identified using the R package SCTransfom (version 0.2.1). Subsequently, principal component analysis was performed, and the appropriate principal components (PCs) for dimensionality reduction were decided using the JackStraw function. Clusters were identified with the Seurat function FindClusters with the resolution set at 0.4. This method resulted in 18 clusters, which were visualized by Uniform Manifold Approximation and Projection (UMAP) analysis. Clusters were then annotated by using the expression of known genes. We annotated cell types based on cell markers and the R package SingleR  (103 (36). SingleR .According to the previously established inclusion criteria, GSE48452, GSE37031, GSE61260, GSE63067 and GSE130970 were included in this study. There are 104 NASH patients and 70 controls in these five datasets. After outlier removal, 97 NASH patients and 67 controls were retained in the following analysis. The detail information of the five datasets was shown in p value < 0.05, absolute difference of mean > 0.3) among control and NASH in MergeCohort were identified, consisting of 600 upregulated and 231 downregulated DEGs  distinguished between NASH and control group is shown in In the present study, we performed GO and Reactome pathway enrichment analysis to determine the potential functions of 831 DEGs in the pathogenesis of NASH. The biological process analysis , and followed by Cyan , Grey60 , Tan  and Magenta , downregulated genes was significantly enriched in Black  and Brown module . To investigate which co-expression modules are associated with NASH status, we then correlated the expression of eigengenes (genes representing the expression profile of each module) with NASH status. The relationship between all the modules and the NASH status are displayed in a correlation heatmap, in which Y-axis corresponds to groups of genes (modules) and the X-axis represents the NASH status (p < 1.47 × 10-3), which is determined based on Bonferroni correction. Among them, nine modules  were positively correlated with NASH disease status, two modules (Black and Brown) were negatively associated with NASH disease status  was conducted based on the top 5000 median absolute deviation (MAD) genes from the MergeCohort. Keep to the scale-free topology criterion, β=5 was considered in this study (p < 0.01), therefore indicating that the modules detected in the present work are biologically relevant     (p = 2.0 × 10-33) between the upregulated fibrosis module Cyan and downregulated Black module that is enriched in metabolic processes  . Interesrocesses , 40.p = 1.3 × 10-9)  (p < 0.05) and none significantly downregulated . The expression level of those core genes were all upregulated in four cohorts  involved in this study Hub genes were upregulated in the liver from NASH patients. Focusing on the MergeCohort_Turquoise module, we firstly explored the top 25 hub genes including FPR3 has the highest correlation . LCP1 gene was the most associated gene with steatosis grade  and the lobular inflammation grade . Moreover, FPR3 associated most with the cytological ballooning grade . SRGN was the most relevant gene with the fibrosis stage . Additionally, C3AR1 showed significant correlation with all the clinical parameters, especially higher correlation with the cytological ballooning grade .Hub genes were positively correlated with clinical characteristics. We further investigated the relationship between the changes in expression of these 25 hub genes and the histological phenotype in GSE130970  model of NASH in mouse. Furthermore, to explore the significance of the hub genes in mouse, we mined public available microarray data (GSE120977)  to validCD53, LCP1, LAPTM5, PTPRC and SRGN expressed by distinct immune cells such as microphages, NK cells, T cells, dendritic cells and B cells, and most of them, namely FGL2, HCK, MPEG1, CYBB, CSF1R, IGSF6, CPVL and HLA-DRA were mainly expressed by macrophages, dendritic cells  , we further conducted TFs enrichment analysis (hypergeometric test) using the genes from the MergeCohort_Turquoise and GSE135251_Turquoise modules to obtain key regulatory genes, based on TRRUST database . Our resRFX5 and ILF3, an important transcriptional factor mainly expressed in the liver, upregulated from mild to advanced NASH, regulates the expression of genes involved in antigen processing and presentation of exogenous peptide antigen via MHC class II, including HLA-DQB2, HLA-DOA, HLA-DMA, HLA-DQA1, HLA-DMB, HLA-DPB1, HLA-DPA1 and HLA-DRA. Notably, the gene expression of RFX5 and ILF3 positively correlated with MHCII gene expression  and immune response   including hub genes (CYBB and HCK) in MergeCohort_Turquoise module. SP1 regulated gene involved in cell activation .Next, the regulatory networks were constructed for the enriched TFs and associated target genes in each of the modules  and murine primary hepatocytes (PRJNA726846) treated with palmitic acid and oleic acid (PAOA) for 0h, 12h and 24h, respectively (CD53 and SRGN) and key TFs  displayed higher expression in L02 hepatocytes treated with PAOA (Lcp1 and Fcer1g) and key TFs  showed increased expression in murine primary hepatocytes with PAOA treatment , we fouThe global epidemic of NASH is a serious public health problem, the pathogenesis of NASH still remains unclear. Moreover, although liver biopsy currently remains the reference standard for diagnosis of NASH, it is an intrusive operation with risks and many shortcomings. Thus, identifying novel non-invasive biomarkers in NASH is of paramount importance in the prevention and therapy of this disease.Thanks to the rapid development of high-throughput sequencing technology and gene chip technology, more and more researchers are actively pursuing molecular markers using data mining and analysis of sequencing data or gene chips to the diagnosis and treatment of disease , 43, 44.FDFT1, NSDHL, IDI1, SQLE, MVD, HMGCS1, HMGCR and LSS) as well as fatty acid metabolism   and fatty acid metabolism genes  in NAFLD mouse model that were also reported to be deregulated by FDFT1, NSCD53, LCP1, LAPTM5, NCKAP1L, C3AR1, FGL2, PLEK, HLA-DRA, FPR3 and SRGN, which also showed positive correlation with histological grade  activation when compared with the wildtype littermates (LCP1 in liver tissue of NAFLD patients was strongly increased (300%) compare to the control group in a previous GWAS study , a member of NF-κB family, emerging evidence suggests that NF-κB1-gene-coded proteins p105 and p50 have critical regulatory activities of inflammatory responses -binding protein which can regulate gene expression and stabilize mRNA  risk genes HLA-DRB1 and HLA-DQA1 expression in human microglial cells  maturation, and found that knockdown of ILF3 led to significantly elevated expression of genes  associated with DC maturation in the primary human monocyte-derived DCs during stimulation with viral mimetics or classic innate agonists (ILF3 divergent transcript (ILF3-AS1) in HCC, Bo et al. found that ILF3-AS1 expression was significantly increased in HCC tissues and also associated with prognosis of HCC patients, and knockdown of ILF3-AS1 expression suppressed HCC cell proliferation, migration and invasion (ILF3-AS1 silencing inhibited the hepatocellular carcinoma tumor growth (RFX5 and ILF3 on HLA-DR molecules in the progression of NASH have also not been well defined. Therefore, our results provide a very meaningful direction for future research.ize mRNA , 94. Recal cells . Moreoveagonists . In addiinvasion . Yan et r growth . HoweverRFX5 coordinating antigen processing and presenting function in NASH progression deserve further attention. The main limitation of present study is that all conclusions are based on transcriptomic data from human and lack verification from relevant experiments in vitro/in vivo disease models. Nevertheless, it provides useful and novel molecular candidates in dysregulated pathways for NASH prognosis and therapeutic targets.In summary, unlike previous studies with limitation of a few human NASH transcriptome data or focusing on individual genes influencing NASH progression, our network-driven strategy generated a comprehensive and unbiased view of the modules, hub genes and critical transcriptional factors associated with NASH. In particular, the Turquoise module and regulators involving immune-related pathways especially transcription factor The original contributions presented in the study are included in the article/Conception and design: J-JZ and FX. Acquisition and analysis of data: J-JZ, YS and X-YC. Investigation: J-JZ, YS, X-YC, M-LJ, F-HY and JZ. Software: J-JZ. Validation: YS, X-YC, M-LJ, S-LX and JZ. Visualization: J-JZ. Writing–original draft: J-JZ. Writing–review & editing: J-JZ, X-YC, F-HY and FX. Funding: J-JZ. All authors contributed to the article and approved the submitted version.


Anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) is a class of small vessel vasculitis that includes granulomatosis with polyangiitis (GPA), eosinophilic GPA (EGPA), and microscopic polyangiitis (MPA). Despite extensive research, the mechanisms behind AAV etiology remain obscure. The genetics of AAV is a complex area of investigation because of the rarity of familial cases. However, recent multi-center genome-wide association studies (GWAS) have greatly contributed to our understanding of the genetic basis of AAV. In this study, we report a rare occurrence of GPA in two Caucasian family members who presented with similar clinical symptoms and performed a comprehensive review to study the present literature available regarding the heritability of this disease. Anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) is a type of small vessel vasculitis characterized by inflammation of both arteries and veins. AAV includes granulomatosis with polyangiitis (GPA), eosinophilic GPA (EGPA), and microscopic polyangiitis (MPA). GPA is a rare necrotizing vasculitis often affecting the upper and lower respiratory tract and the kidneys. Diagnosis is based on characteristic clinical and pathological findings, usually in the presence of ANCA directed against neutrophil and monocyte proteinase 3 (PR3) or myeloperoxidase (MPO) ,2.Despite extensive research, the mechanism behind AAV etiology remains obscure . There sWhile familial aggregation has been reported in other autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, there have been very few cases of GPA clustering in families. It has been shown that these family members shared an identical HLA haplotype, i.e., HLADPB1 allele, which has been linked to GPA in previous studies as well ,6.Case 1The patient was a 62-year-old female who presented initially in 2018 with the complaint of bilateral lower extremity pain, ulcerations, erythema, and swelling. She had been experiencing polyarthralgia mainly involving small joints of the fingers, scattered petechial/purpuric rash on the upper and lower extremities, and black discoloration of fingertips. She was started on methotrexate by an outpatient rheumatologist for suspected inflammatory arthritis and had a pending autoimmune workup.On presentation, she was vitally stable. A physical exam revealed black discoloration with areas of necrosis on the fingertips. She had blanchable erythema on the hands and feet, with areas of necrosis on the tips of her toes Figure . The patInvestigationsLaboratory investigation showed an elevated WBC count of 12.6 x 10*3/uL  and low hemoglobin of 10.8 g/dL . Renal function and urinalysis were normal. Erythrocyte sedimentation rate (ESR) was elevated at 60 mm/hr . Serology showed elevated titers of antinuclear antibody (ANA) 160 with indirect immunofluorescence showing a speckled pattern , cytoplasmic ANCA of 320 , and anti-proteinase-3 of 51.3 . Rheumatoid factor was elevated to 62 IU/ml . Perinuclear ANCA, atypical ANCA, and anti-MPO were normal. An autoimmune panel including anti-centromere Ab, anti-RNA polymerase III Ab, anti-Scl-70 Ab, Sjogren's panel including anti-SSA/SSB, anti-phospholipid Ab, anti-cardiolipin Ab, anti-Beta-2 glycoprotein Ab, anti-CCP Ab, anti-glomerular Ab were negative. The patient had normal complement levels and negative cryoglobulins. HIV and hepatitis B/C screens were negative , elevated ESR 64 , and CRP 156 . Renal function was normal. Urinalysis showed 1+ protein, 1+ leukocyte esterase, 3+ hemoglobin, and 10-20 RBCs per hpf. Chest x-ray showed bilateral nodular opacities in mid and lower lung fields. This was followed by a CTA chest which showed centrilobular nodular opacities in bilateral lung fields concerning intra-alveolar bleeding. Rheumatology was consulted due to concerns about an autoimmune/vasculitic process. The patient was started on oral prednisone, pending further workup  and anti-proteinase-3 was 375.1 . Rheumatoid factor was elevated to 108 . Hepatitis B and C panels were negative.Bronchoalveolar lavage was done in the setting of worsening hemoptysis which retrieved bloody fluid; however, cultures were negative. Skin punch biopsy hematoxylin and eosin stained sections showed superficial to deep dermal perivascular infiltrate of neutrophils, occasional eosinophils, karyorrhexis, and red blood cell extravasation with a prominent fibrinoid change of vessel wall. Direct immunofluorescence showed deposition of IgM and C3, C5b, and C4d with some fibrinogen in superficial dermal vessels consistent with leukocytoclastic vasculitis Figure .In light of these findings  and family history of GPA, the patient was diagnosed with an autoimmune-mediated small vessel vasculitis, with the leading differential being GPA. HUV was ruled out in the setting of normal complement levels , negative ANA, and Sjogren's panel. Others on the differential, but less likely include EGPA which was ruled out in the absence of eosinophilia, normal IgE levels, negative p-ANCA/anti-MPO Ab, or clinical features like asthma and neuropathy. Cryoglobulinemic vasculitis was ruled out in the absence of cryoglobulins.TreatmentThe patient was started on a pulse dose of steroids. He had worsening hypoxic respiratory failure, requiring intubation. The plan was to initiate rituximab or cyclophosphamide along with plasmapheresis. However, the patient quickly deteriorated in the ICU. He went into sustained ventricular tachycardia followed by pulseless electrical activity arrest and died on the sixth day of hospitalization despite aggressive resuscitation efforts.Literature reviewMethodsIn this study, we report a rare occurrence of GPA in two Caucasian family members, a mother and her son who presented with similar clinical symptoms and performed a comprehensive literature review.A literature search was conducted on PubMed, Cochrane, and Google Scholar from inception to December 2022 using Medical Subject Headings (MeSH) terms for "granulomatosis with polyangiitis," "ANCA vasculitis," and "familial." The search strategy combined free text search, MeSH terms, and synonyms for identifying observational studies, case series, and case reports. We also searched for additional articles from the reference list of the original articles.Eligibility CriteriaWe included case reports, case series, and observational studies which reported the familial cases of GPA.Study SelectionThe authors screened the titles and abstracts of all references which were identified by the literature search and excluded irrelevant studies. We read the full texts of the remaining articles and included the studies which met the inclusion criteria.Data ExtractionThe authors extracted data from the included studies using a pre-specified data collection form in Microsoft Excel. Data about the study year, patient characteristics, serological profile, diagnosis, treatment, and outcome were extracted.ResultsA total of 8 studies and 17 patients were included. The mean age was 39.2 years, sex distribution was 58.8% (n=10) males and 41.1% (n=7) females. About 88.2% of the cases were diagnosed with GPA (n=15); however, EGPA was found in 5.8% (n=1) and MPA in 5.8% (n=1) of the familial cases. Serological results were diverse, and out of 13 patients, 38.4% (n=5) were c-ANCA positive, 23% (n=3) had p-ANCA, 23% (n=3) had anti-MPO Ab, and 30 % had anti-PR3 Ab(n=4). Twenty-three percent of the patients were positive for ANCA (n=3) without specifying whether c-ANCA or p-ANCA. Serological results were not reported for 4/17 patients. Out of the total 17 patients, 15 received treatment. About 93.3% (n=14) were treated with steroids, 86.6% (n=13) received cyclophosphamide, 53.3% (n=8) received azathioprine, 13.3% (n=2) received rituximab, 6.6% (n=1) received methotrexate, and 6.6% (n=1) received plasmapheresis. After the treatment phase, all patients survived, 35.3% (n=6) went into remission, while 17.64 % relapsed (n=3)  have been performed to identify the genetic factors associated with AAV. The first GWAS in a European population in 2012 demonstrated that HLA-DP rs3117242 (G) was the strongest signal in the HLA region .Another GWAS for GPA with 492 patients and 1,506 healthy individuals of European descent identified 32 SNPs across the HLA region; among them, HLA-DPB1 rs9277554 and HLA-DPA1 rs9277341 were significantly associated with GPA. In another study with 150 GPA patients and 100 healthy controls conducted in northern Germany, HLA-DPB1*0401 was identified to be associated with GPA, and DPB1*0401/RXRB03 haplotype frequency was significantly increased in patients with GPA ,20. GWASIt is crucial to acknowledge that a subset of patients with GPA may test negative for ANCA. These ANCA-negative cases are more commonly observed among individuals with limited disease. Therefore, it is imperative to thoroughly review the complete classification criteria before reaching a final diagnosis .The etiology behind the mechanics of AAV, specifically GPA, remains obscure; however, recent studies and case reports have predicted a genetic component to play a pivotal role. Genetic testing, HLA subtyping, and further studies are warranted to determine the possible hereditary transmission of the disease.In summary, these findings could help elucidate the etiology of AAV and develop new biomarkers for early diagnosis and targeted therapy. Although very informative, all these findings represent only the beginning of a new exciting, and dynamic phase in this field. Furthermore, information on the possible heritability of AAV is of clinical importance because family members would often want to know whether having AAV puts their closest relatives at increased risk of developing the disease.


Remarkably, utilizing mild reaction conditions, a wide range of aryl halides, including fluorides, chlorides, bromides, and iodides, can be selectively transformed into their corresponding (hetero)arene counterparts, with exceptional yields. Additionally, this approach demonstrates a remarkable compatibility with diverse functional groups and heterocyclic compounds, highlighting its versatility and potential for use in various chemical transformations.In this study, we present a straightforward and highly effective photo-triggered hydrogenation method for aryl halides, devoid of transition-metal catalysts. Through the synergistic utilization of light, PhNHNH Reductive hydrodehalogenation, an important chemical transformation, finds extensive applications in environmental protection, medicinal chemistry, and organic synthesis. This process plays a critical role in modifying bioactive molecules with halogen groups during the late stages, enabling precise structural adjustment. Moreover, it assumes a vital role in converting hazardous and long-lasting halogenated organic pollutants into eco-friendly compounds ,6,7,8,9.2, hydrosilanes, hydrides, and alcohols, are extensively applied in this context. Furthermore, scholars have explored the dehalogenation of aryl or alkyl halides through electrolysis, wherein a metal electrode is employed to provide electrons. They have also studied photo-induced dehalogenation, which involves using amines as terminal reductants and hydrogen atom donors, in combination with organic or inorganic photocatalysts and various additives [To address the significance of hydrodehalogenation, organic chemists have dedicated considerable efforts over the decades to developing efficient and straightforward methods. Traditional methods typically employ AIBN as an initiator for the radical reductive dehalogenation of aryl halides. Additionally, transition-metal-catalyzed reductions using diverse agents, such as Hdditives ,20,21,22However, these approaches are accompanied by inherent constraints, such as an inadequate selectivity resulting in diminished yields, rigorous reaction conditions, dependency on costly noble metals, the necessity of inert atmospheres and specific ligands, an excessive utilization of potentially hazardous radical initiators, and the requirement for specialized photo/electrochemical reactors. Therefore, there is significant interest in developing a straightforward and highly efficient dehalogenation method that utilizes light as a trigger, aiming to avoid the use of expensive metal catalysts, complex additives, and specialized experimental setups ,36,37,38t-BuOK and eight equivalents of Et3N, while exposing the mixture to blue LED light , yielded only 6% of the dechlorinated product 2a (entry 1). However, the introduction of PhNHNH2 into the system significantly improved the dichlorination outcome (entries 2–5). To explore the impact of different solvents on the dichlorination process, various solvents, including MeCN, 1,4-Dioxane, and t-BuOH, were evaluated (entries 6–8), resulting in dichlorination yields ranging from 15% to 38%. Different light sources, such as 254 nm, 395 nm, 405 nm, 455 nm, and 485 nm, and even the absence of light (dark condition), were also examined, but none surpassed the efficiency of 365 nm blue LED irradiation (entries 9–14). Furthermore, investigations into the reaction without t-BuOK or Et3N present were conducted, which led to 8% and 26% yields of 2a, respectively. To optimize the reaction conditions further, different base combinations were tested (entries 15–19), among which 8 eq. of t-BuOK in combination with 16 eq. of N,N′-Dimethyl-1,2-ethanediamine proved to be the most efficient. The effects of lowering the reaction temperature were also evaluated (entry 20), but this resulted in a decrease in the dichlorination yield. After a comprehensive comparison of all the conditions in An exploration of reaction conditions was conducted, employing the dehalogenation of 4-chloro-1,1′-biphenyl 1a as a representative model reaction. A comprehensive summary of the findings is presented in Having established the optimized conditions, we extensively investigated the reactivity of diverse aryl chlorides . Under sThe hydrodefluorination of organic fluorides represents a valuable strategy, not only for detoxifying environmentally hazardous halogenated chemicals but also for its significant potential in molecular functionalization, especially in the field of drug discovery. Despite the rapid progress in hydrodefluorination methodologies, the light-promoted hydrodefluorination of aryl fluorides remains relatively scarce. However, upon subjecting aryl fluorides 3a–d to the specified standard conditions outlined in In order to comprehensively explore the applicability of this system, we conducted additional investigations involving aryl bromide 4 and aryl iodide 5. The outcomes of these experiments are succinctly presented in To elucidate the underlying mechanism governing this dehalogenation reaction, a series of insightful control experiments was meticulously conducted. The addition of 1 equivalent of the standard radical scavenger TEMPO led to a reduction in the yield of 2a under the specified conditions.Furthermore, a progressive decrease in the yield of 2a was observed with an increased amount of TEMPO, eventually leading to the complete inhibition of the reaction upon the introduction of TEMPO . Moreove2 (A), which donates an electron to aryl halide 1 under the influence of LED light. This electron transfer results in the formation of radical anion C and radical B. Subsequently, aryl radical D captures a hydrogen atom from DMF, yielding product 2 and releasing E. E undergoes attack from t-BuOK, forming radical anion F. It is worth noting that radical anion F plays a crucial role as the principal electron donor in the subsequent stages of the reaction [Based on the insights gleaned from the radical trapping experiments, we propose a plausible mechanism, which is illustrated and substantiated in reaction ,30,31,322, LED light, and a suitable base system. Notably, a wide range of aryl halides efficiently underwent reduction, yielding the corresponding products in yields ranging from moderate to excellent, all under mild reaction conditions. The method exhibits a remarkable tolerance toward various functional groups and heterocyclic compounds, underscoring its practicality in synthetic applications.In this study, we introduce a novel and straightforward strategy for the transition-metal-free, light-driven hydrogenation of aryl halides. The key to initiating this radical-mediated hydrogenation lies in the synergistic use of PhNHNH3. The determinations of some chemicals were compared with the spectra reported in the literature. The light sources employed were LED lamps with wavelengths ranging from 254 nm to 485 nm, providing an output power of 7 W. The reaction progress was monitored via thin-layer chromatography (TLC). Column chromatography was performed using silica gel (200–300 mesh), and the compounds were visualized under ultraviolet light. Elution during column chromatography was achieved using a mixture of ethyl acetate and petroleum ether as the eluent. High-performance liquid chromatography (HPLC) analyses were conducted using a Shimadzu LC-16 spectrometer. Gas-chromatography–mass-spectrometry (GC-MS) analyses were carried out utilizing a Thermo TRACE 1300 ISQ LT spectrometer.All reagents and solvents utilized in this study were procured from commercial sources and employed without further purification, unless otherwise specified. Nuclear magnetic resonance (NMR) spectra were acquired using Bruker AV300, Bruker AV400, and Bruker AV500M spectrometers, and chemical shifts were reported in parts per million (δ) relative to the internal standard, tetramethylsilane (TMS), positioned at 0 ppm in CDClt-BuOK (1.6 mmol), PhNHNH2 (0.8 mmol), N,N’-Dimethyl-1,2-ethanediamine (3.2 mmol), and dry DMF (3 mL). Subsequently, the pressure vessel was immersed in an oil bath along with LED lamps  at a temperature of 50 °C. To ensure light exclusion, the entire reaction system was carefully shielded with tinfoil film. The reaction mixture was stirred for 48 h. Following the designated reaction time, 0.2 mmol of 4-methylbiphenyl was introduced into the pressure vessel. Subsequently, 10 mL of water was added to terminate the reaction, and the resulting mixture was extracted using ethyl acetate (5 mL × 4). The combined organic extracts were washed with brine, dried with sodium sulfate, and then subjected to filtration for the subsequent HPLC analysis.A 35 mL thick-walled pressure vessel equipped with a Teflon cap was charged with a magnetic stirring bar, 4-chloro-1,1’-biphenyl (0.2 mmol), t-BuOK (1.6 mmol), PhNHNH2 (0.8 mmol), N,N’-Dimethyl-1,2-ethanediamine (3.2 mmol), and dry DMF (3 mL). The pressure vessel was then positioned in an oil bath, accompanied by LED lamps emitting light at a wavelength of 365 nm and a power output of 7 W × 2, while maintaining a temperature of 50 °C. To ensure light exclusion, the entire reaction system was meticulously wrapped with tinfoil film. The mixture was stirred for 48 or 96 h, as specified. Following the designated reaction time, 10 mL of water was added to quench the reaction, and the resulting mixture was extracted using ethyl acetate (5 mL × 4). The combined organic extracts were subsequently washed with brine, dried with sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified via preparative TLC on silica gel, using a gradient elution of petroleum ether and ethyl acetate (300:1–5:1) as the eluent, to yield the desired products. The confirmation of the products was accomplished via comparison with commercially available samples ,  samples .