Drosophila melanogaster olfactory neurons have long been thought to express only one chemosensory receptor gene family. There are two main olfactory receptor gene families in Drosophila, the odorant receptors (ORs) and the ionotropic receptors (IRs). The dozens of odorant-binding receptors in each family require at least one co-receptor gene in order to function: Orco for ORs, and Ir25a, Ir8a, and Ir76b for IRs. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the main chemosensory families in D. melanogaster . Co-receptor knock-in expression patterns were verified as accurate representations of endogenous expression. We find extensive overlap in expression among the different co-receptors. As defined by innervation into antennal lobe glomeruli, Ir25a is broadly expressed in 88% of all olfactory sensory neuron classes and is co-expressed in 82% of Orco+ neuron classes, including all neuron classes in the maxillary palp. Orco, Ir8a, and Ir76b expression patterns are also more expansive than previously assumed. Single sensillum recordings from Orco-expressing Ir25a mutant antennal and palpal neurons identify changes in olfactory responses. We also find co-expression of Orco and Ir25a in Drosophila sechellia and Anopheles coluzzii olfactory neurons. These results suggest that co-expression of chemosensory receptors is common in insect olfactory neurons. Together, our data present the first comprehensive map of chemosensory co-receptor expression and reveal their unexpected widespread co-expression in the fly olfactory system. The sense of smell is crucial for many animal behaviors, from conspecific recognition and mate choice , to locaDrosophila melanogaster, is one of the most extensively studied and well understood , complex olfactory-driven behaviors, and similar organizational principles to vertebrate olfactory systems , ionotropic receptors (IRs), and gustatory receptors (GRs) , and all neurons expressing the same receptor (which can be considered an OSN class) project their axons to one specific region in the AL called a glomerulus (IR (Ir76b) in addition to the OR and Orco, and innervates the VC3 glomerulus  . These rrs (GRs) , to the rs (GRs) . The staomerulus . This paomerulus , teal, Iomerulus , purple,omerulus . Differeomerulus .ORs require a single co-receptor, Orco, to function  are similarly required for trafficking and ion channel function   . The ORsfunction . The lig channel . Likewisor Ir76b , top rowfunction . The GR he legs) ; however dioxide .Ir25a-Gal4 line is known to label only a portion of cells expressing Ir25a protein   ; conversomerulus . While kD. melanogaster . We find broad co-expression of these co-receptor genes in various combinations in olfactory neurons, challenging the current view of segregated olfactory families in the fly. In particular, Ir25a is expressed in the majority of olfactory neurons, including most Orco+ OSNs. In addition, the Ir8a and Ir25a knock-in lines help to distinguish two new OSN classes in the sacculus that target previously unidentified glomerular subdivisions in the posterior AL. Recordings in Ir25a mutant sensilla in Orco+ neurons reveal subtle changes in odor responses, suggesting that multiple chemoreceptor gene families could be involved in the signaling or development of a given OSN class. We further extend our findings of co-receptor co-expression to two additional insect species, Drosophila sechellia and Anopheles coluzzii. These data invite a re-examination of odor coding in D. melanogaster and other insects. We present a comprehensive model of co-receptor expression in D. melanogaster, which will inform future investigations of combinatorial chemosensory processing.In this paper, we implement an efficient knock-in strategy to target the four main chemosensory co-receptor genes in Gal4 to QF2 , legs, and wings  showed innervation in the VNC, consistent with the role of these genes in gustation in addition to olfaction , corresponding to gustatory axons from the labella  . Only twlfaction . In the lfaction , green tal4 line , orange  labella . The co-Orco-T2A-QF2 labels seven ‘non-canonical’ glomeruli consistently, and one sporadically. These include VM4 and VL2a, which correspond to Ir76b+ and Ir8a+ OSN populations, respectively , three additional Ir8a+ glomeruli consistently , and one variably (DC4). Surprisingly, when we crossed the transgenic Orco-Gal4 line .al4 line  to a stral4 line , we founal4 line . This suIr8a-Gal4 line labels approximately 10 glomeruli, 6 of which are identified . An Ir8a-Gal4 line generated by Ir8a-Gal4 line  but not reported in the other two papers. DL2 was later subdivided into two glomeruli  , most Ir8a+ glomeruli (including DP1l), and one additional Ir25a+ glomerulus (VL1)  . The Ir7us (VL1) . As withreporter . Howevervariably .Ir25a-T2A-QF2 innervation of the AL was the most expanded compared to what has previously been reported. In addition to the novel expression we identified in the palps , it was assumed that neurons not captured by the transgenic line would reside in coeloconic sensilla, the arista, or sacculus  , immunohistochemistry, and optogenetics.he palps , we founantennae . The extR+ OSNs) . When welomeruli , but thiD. melanogaster and suggests two possible explanations: either the same olfactory neurons express multiple co-receptors (co-expression) or different populations of olfactory neurons expressing different receptors converge upon the same glomeruli (co-convergence). These scenarios are not necessarily mutually exclusive. To examine these possibilities in a comprehensive, unbiased way, we analyzed snRNAseq data from adult fly antennae . Similarly, Ir8a and Ir76b are expressed in cluster 19 , and Ir25a is expressed in multiple Orco+ clusters .The innervation of the same glomeruli by multiple co-receptor knock-in lines challenges the previous view of segregated chemosensory receptor expression in antennae . Figure Ir25a-T2A-QF2>GFP antennae and palps , a necessary co-factor for channelrhodopsin function . We used similar optogenetic experiments to examine Ir25a expression in OSN classes innervating DM4  and DM5  (Ir25a is expressed in ab2A (DM4) and ab9B (VA3) neurons, but not ab2B (DM5) or ab9A (D) neurons  , as well/Or33b+) . These esee also . ResultsIr8a-T2A-QF2 and Ir25a-T2A-QF2 knock-ins strongly labeled a previously uncharacterized posterior region of the AL. By performing a co-labeling experiment with Ir41a-Gal4, which labels the VC5 glomerulus, we narrowed down the anatomical location of this region and ruled out VC5 as the target of these axons  and Ir8a protein (narrow overlap) in the third chamber of the sacculus ; Ir8a-T2A-QF2 consistently labels 18 glomeruli (8 more than previously identified); Ir76b-T2A-QF2 consistently labels 15 glomeruli (11 more than previously identified); and Ir25a-T2A-QF2 consistently labels 51 glomeruli (39 more than previously identified).Orco and Ir25a). We chose to test eight OSN classes previously assigned to the Orco+ domain that we found to have strong or intermediate Ir25a expression – two in the antennae and six in the maxillary palps. The two antennal OSN classes are found in the same ab3 sensillum . The six palpal OSN classes represent the entire known olfactory neuron population of the maxillary palps . In both the antennae and the palps, we compared the olfactory responses of OSNs to a panel of 13 odorants in three genotypes: wildtype, 2Ir25a mutant, and 2Orco mutant flies. This panel included odorants typically detected by ORs, such as esters and aromatics, and odorants typically detected by IRs, such as acids and amines  Ir25a is not expressed in these neurons or (b) Ir25a is expressed, but is not playing a functional role in these neurons. In an 2Orco mutant background, there would be no trafficking of Orco/OrX receptors to the dendritic membrane, and no formation of functional ion channels  contain two neurons each (A and B) , for a tR ligand , and IR 2 mutant . This way ~6 DPE , which c neurons . There we mutant . This su2Ir25a mutant compared to the wildtype , as was the response to methyl salicylate (p=0.0177), while the response to ethyl acetate (EA) was higher in wildtype (p=0.008)  in pb3B. Interestingly, when we examined a list of candidate IrX tuning receptors  in the 2Orco mutant; however, across all flies tested, these responses were not significantly different from the mineral oil control , 1-octen-3-ol (p=0.0004), phenylacetaldehyde (p=0.015), and pentyl acetate (p=0.0164). Interestingly, two of these four odorants are typically associated with IRs . In the ab3B neuron, only two odorants elicited significantly different responses between the wildtype and 2Ir25a mutant: propionic acid , and pentyl acetate . While responses to propionic acid are small in both ab3 neurons, they are abolished in the 2Ir25a mutant background , suggesting that propionic acid detection in ab3 may be Ir25a-dependent.As in the palps, we found significant differences in the responses of both ab3A and ab3B neurons to some odorants between the two genotypes. A comparison of all ab3A responses between the D. melanogaster, we used RNA in situ hybridization to examine expression of Orco and Ir25a orthologues in the fly D. sechellia and in the mosquito A. coluzzii , consistent with our findings in D. melanogaster , and narrower co-expression in the antennae (25% double labeled). Co-expression results for all tissues examined are summarized in Orco and Ir25a co-receptor co-expression extends to other Drosophilid species as well as mosquitoes , and glomeruli not labeled by the given knock-in are in white. The new VM6v, VM6m, and VM6l subdivisions are labeled with gray stripes.Co-receptor co-expression of insect chemosensory receptors suggests that multiple receptors may influence the response properties of an olfactory neuron, as we have shown in ab3 and palpal sensilla. To aid future investigations of co-receptor co-expression signaling, we synthesized our results  into a cDrosophila, the Orco/OR domain primarily occupied the anterior AL, while the IR domains innervated more posterior glomeruli. While the former is still, for the most part, accurate (Orco), the latter is not: both Ir8a-T2A-QF2 and Ir76b-T2A-QF2 label several more anterior glomeruli (such as VA3 or VA6), and Ir25a-T2A-QF2 labels the majority of glomeruli throughout the anterior to posterior axis (Ir25a). The expansion of the Ir25a+ domain is the most dramatic of the four co-receptors: previously, Ir25a+ glomeruli accounted for 21% of the AL (12/58 glomeruli) . This represents a greater than fourfold expansion. Similarly, the number of Ir76b+ glomeruli increased more than threefold, from 7% of the AL (4/58 glomeruli)  ; the Ir2omeruli)  to 26% (omeruli)  to 31% (omeruli)  to 78%  might also be more broadly expressed than previous transgenic lines indicate  (Orco and IrCo(s)   is shown in indicate . All fouOrco-T2A-QF2 knock-in , and neurons that express Gr21a/Gr63a, Ir25a, and Orco   co-converging onto the same glomerulus. There is some evidence for this in the palps, based on our anti-Orco and anti-Ir25a antibody staining , we demonstrate that all four co-receptors have broader olfactory neuron expression than previously appreciated. The Ir25a co-receptor was previously thought to be expressed only in a small subset of olfactory neurons , but we present evidence that it is expressed in subsets of all sensilla classes and in OSNs that innervate the majority of the fly’s AL glomeruli. We further find that the Ir25a co-receptor may be involved in modulating the activity of some Orco+ OSNs, both in the antennae and maxillary palps. We present a new AL map that will aid future inquiries into the role that specific chemoreceptor co-expression plays in distinct OSN populations.Here, we present evidence of widespread chemosensory co-receptor co-expression in the olfactory system of Ir25a-T2A-QF2 and Ir8a-T2A-QF2 knock-in lines, the cell bodies of these neurons had to reside in the antenna; furthermore, since we did not find Ir8a-T2A-QF2 labeling of the arista, these neurons were likely to be either in coeloconic sensilla or in the sacculus. Indeed, we determined the VM6 glomerulus to be innervated by the newly discovered Rh50+ Amt+ olfactory neurons that reside in the sacculus and ac1 sensilla  are often weakly or stochastically labeled. This might also explain why Ir25a expression was initially overlooked in these Orco+ neural populations. The developmental pattern is different in the maxillary palps, where it is atonal and not amos, which is required for the development of the basiconic sensilla  : thus, molecularly, the VM4 neuron profile is more similar to DP1m  than DC4 (co-expressing two IrCos), and the key distinguishing component appears to be Ir76b. It would be interesting to repeat such misexpression experiments in an -Ir76b mutant background to test this hypothesis.Chemosensory co-receptor co-expression may help clarify previously confounding observations regarding and DP1m . These trco) see . In conte to VM4 . We showIr25a plays a functional role in Orco+ neurons in the antennae and palps; this suggests that these Orco+ neurons could also express as yet unidentified ligand binding IrXs. The recent release of the whole fly single-cell atlas, which includes RNAseq data from maxillary palps, allowed us to identify six IRs that might be expressed in palpal OSNs   that elicited a small response in wildtype neurons and no response in 2Ir25a mutant neurons. It is possible that other antennal Orco+ OSNs might utilize IR chemoreceptors for signaling. For example, the ac3B neuron, which expresses Or35a/Orco and all IR co-receptors, has recently been suggested to utilize an unidentified IrX to mediate responses to phenethylamine  receptors. Previous studies suggest that OrX tuning receptors are generally limited to a single class of olfactory neurons . However, Ir93a) . Howeverhylamine . The cheIr25a in the fly olfactory system raises the possibility that it might have roles in addition to its function as an IrX co-receptor. For example, Ir25a has been found to play a developmental role in forming the unique structure of Cold Cells in the arista .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Christopher J. Potter (Fly lines generated in this study have been deposited to the Bloomington Drosophila Stock Center.https://github.com/colleen-mclaughlin/ORN_seq/). VM6 reconstructions using FlyWire can be viewed at https://flywire.ai/#links/Task2021a/all. Raw data used to generate each figure is available at Johns Hopkins Research Data Repository (https://doi.org/10.7281/T1/9VJGPI ).The snRNAseq dataset analyzed in this paper is published in Fly stocks were maintained at 20–25°C on standard cornmeal-agar food. Male and female flies used for experiments were 3–11 days old, unless otherwise noted.Fly lines used in this paper can be found in the Appendix 1—key resources table.OrX-Gal4 lines label multiple glomeruli, and thus do not accurately represent single OSN classes. These lines were excluded from analyses and should be used with caution: Or33c-Gal4 (BDSC# 9966), Or42a-Gal4 (BDSC# 9970), Or43b-Gal4 (BDSC# 23894), Or59b-Gal4 (BDSC# 23897), Or65a-Gal4 (BDSC# 9994), Or85a-Gal4 (BDSC# 23133), and Or85b-Gal4 (BDSC# 23911). We also found that the following Or35a lines label the newly identified VM6l glomerulus in addition to VC3: Or35a-Gal4 (BDSC# 9967), Or35a-Gal4 (BDSC# 9968), Or35a-mCD8.GFP (BDSC# 52624), as well as an Or35a-Gal4 line from the Carlson lab  were obtained from the National Drosophila Species Stock Center (Cornell College of Agriculture and Life Sciences) and reared according to our D. melanogaster protocol.A. gambiae mosquitoes were reared as previously described . A fly line was established through random P-element insertion. Cloning was confirmed with Sanger sequencing (Genewiz) before being sent for injection . Primers used for PCR amplification and In-Fusion cloning:The sequence of CsChrimson.Venus was PCR amplified from the genomic DNA of us flies  and clonIVS-FOR:TGGGTTGGACTCAGGGAATAGATCTAAAAGGTAGGTTCAACCACTEcoRI-SV40-REV:GCTTACGTCAGAATTCAGATCGATCCAGACATGATAAGAgRNAs specific to the target gene, as well as the template for HDR-mediated insertion of T2A-QF2 into the genome . We found no difference in expression pattern in the brain between these two approaches (Cre recombination (Orco and Ir25a) and confirmed no difference in brain GFP expression between marked and unmarked lines , we found efficiency rates comparable between approaches . For the direct injection approach, we tested 51 independent knock-in lines across the four target genes and found 100% to be correctly targeted events . For HACKing via genetic cross, Orco-T2A-QF2 and Ir25a-T2A-QF2 constructs were injected into 1118w flies for P-element insertion, and donor lines were established on the second or third chromosomes by crossing to double balancers (see Appendix 1—key resources table). Donor lines were then crossed to Vas-Cas9 (BDSC# 51323). Knock-in lines were established from cis-chromosomal HACK (donor line on same chromosome as target gene) (Vas-Cas9 (BDSC# 51324) for Ir8a; Act5C-Cas9 (BDSC# 54590) for Orco, Ir76b, and Ir25a. The following lines were used to verify knock-in expression: QUAS-mCD8::GFP (BDSC# 30003), 10XQUAS-6XGFP (BDSC# 52264). Knock-in lines were confirmed by PCR genotyping  and Sanger sequencing (Genewiz). Unmarked Orco-T2A-QF2 and Ir25a-T2A-QF2 knock-in lines were generated by crossing mCherry+ knock-in flies to the Crey+ 1B fly line  . For HACT2A-QF2 knock-in on gene function, we performed SSR on homozygous flies for each co-receptor knock-in  as the backbone. The backbone was digested with the following enzymes for cloning: MluI for the 5′ homology arms; SpeI for the 3′ homology arms; and BbsI for the gRNAs. All cloning was performed using In-Fusion Cloning (Clontech #639645). The homology arms were PCR-amplified from genomic DNA extracted from wildtype flies using the DNeasy Blood and Tissue Kit (QIAGEN #69506), while the gRNAs were PCR-amplified using the pHACK-QF2 backbone as a template, with the primers themselves containing the gRNA target sequences. All homology arms were approximately 1 kb . gRNAs were selected by analyzing the region around the stop codon of each gene using an online tool . They were selected such that one gRNA targeted upstream of the stop codon, within the last exon of the gene; the second gRNA targeted downstream of the stop codon, within the 3′UTR; and the two gRNAs were <100 bp apart. In order to prevent the gRNAs from targeting the homology arms, three synonymous nucleotide substitutions were made in each homology arm. The final knock-in lines did not always have all three substitutions GCACAGTGCGGAGGGGGCAA (GGG)Ir8a:GTTTGTTTGTTCGGCCATGT (TGG)GGTGCCTCTGACTCCCACAG (TGG)Ir76b:GCAGTGATGCGAACTTCATA (TGG)GTATTGAAAGAGGGCCGCCG (AGG)Ir25a:GCCGGATACTGATTAAAGCG (CGG)ATTATGGTAAAATGAGCACT (CGG)Italics = In-Fusion Cloning 15 bp overhang; bold = gRNA; lowercase = adding back restriction site; underline = synonymous substitution to prevent Cas9 targeting of donor construct.Orco_gRNA_FOR:TCCGGGTGAACTTCGCACAGTGCGGAGGGGGCAAGTTTTAGAGCTAGAAATAGCAAGTTAOrco_gRNA_REV:TTCTAGCTCTAAAACACTTTATGGTGCTGGTGCAGCGACGTTAAATTGAAAATAGGTCOrco_5HA_FOR:CCCTTACGTAACGCGtCAGCTTGTTTGACTTACTTGATTACOrco_5HA_REV:CGCGGCCCTCACGCGtCTTGAGCTGTACAAGTACCATAAAGTOrco_3HA_FOR:GTTATAGATCACTAGtCTCAGTACTATGCAACCAGCAATAOrco_3HA_REV:AATTCAGATCACTAGtGTTTTATGAAAGCTGCAAGAAATAAIr8a_gRNA_FOR:TCCGGGTGAACTTCGTTTGTTTGTTCGGCCATGTGTTTTAGAGCTAGAAATAGCAAGTTAIr8a_gRNA_REV:TTCTAGCTCTAAAACCTGTGGGAGTCAGAGGCACCGACGTTAAATTGAAAATAGGTCIr8a_5HA_FOR:CCCTTACGTAACGCGtCTATTGGCTATTCGTCGTACTCATGCIr8a_5HA_REV:CGCGGCCCTCACGCGtCTCCATGTAGCCACTATGTGAGTCAGATIr8a_3HA_FOR:GTTATAGATCACTAGtGTTTCTTGTCGCACCTAATTAACAAGTGIr8a_3HA_REV:AATTCAGATCACTAGtCATACTTAAGCTCCTTGAGGTCCAGCIr76b_gRNA_FOR:TCCGGGTGAACTTCGCAGTGATGCGAACTTCATAGTTTTAGAGCTAGAAATAGCAAGTTAIr76b_gRNA_REV:TTCTAGCTCTAAAACCGGCGGCCCTCTTTCAATACGACGTTAAATTGAAAATAGGTCIr76b_5HA_FOR:CCCTTACGTAACGCGtACCAATGAATCCTTGTCCATGCTAAAIr76b_5HA_REV:CGCGGCCCTCACGCGtCTCGGTGTAGCTGTCTTGAAGGAAIr76b_3HA_FOR:GTTATAGATCACTAGtGCCTAATTGGAATACCTTCTACATAATGGAIr76b_3HA_REV:AATTCAGATCACTAGtGGCAAGGCACAAAATAAAACGAAGIr25a_gRNA_FOR:TCCGGGTGAACTTCGCCGGATACTGATTAAAGCGGTTTTAGAGCTAGAAATAGCAAGTTAIr25a_gRNA_REV:TTCTAGCTCTAAAACAGTGCTCATTTTACCATAATCGACGTTAAATTGAAAATAGGTCIr25a_5HA_FOR:CCCTTACGTAACGCGtTGCATGACTTCATTGACACCTCAAGIr25a_5HA_REV:CGCGGCCCTCACGCGtGAAACGAGGCTTAAACGTAGCTGGATATTIr25a_3HA_FOR:GTTATAGATCACTAGtAATATTATGGTTAAGTGAGCTCTCGGIr25a_3HA_REV:AATTCAGATCACTAGtCAAAGCTAAGTTCATCGTCATAGAGACGenotyping and sequencing primers (PCR fragment size):Orco_Seq_FOR (~2 kb):GATGTTCTGCTCTTGGCTGATATTCIr8a_Seq_FOR (~1.9 kb):CATCGACTTCATCATCAGGCTTTCGIr76b_Seq_FOR (~1.9 kb):CAACGATATCCTCACGAAGAACAAGCIr25a_Seq_FOR (~1.9 kb):CGAAAGGATACAAAGGATACTGCATHACK_Seq_REV :TGTATTCCGTCGCATTTCTCTCIr25a-HACKQF2 had one predicted potential off-target cut site in the genome, in the tetraspanin 42ej (Tsp42Ej) gene. We sequenced this locus in the Ir25a-T2A-QF2 knock-in line and compared the sequence to our wildtype lab stock. We found no evidence of indels in the knock-in line. Primers used:One of the gRNAs for Tsp42Ej_FOR:GAGAAGTCGTTTCCCATAACACCCTTsp42Ej_REV:GAGGAGCAGTTTTCGGAGTCGCCTTC2 pad and screened in one of two ways: either with a Nightsea Stereo Microscope Fluorescence Adapter with the green SFA-GR LED light source  and viewed with a Zeiss Stemi SV6 stereo microscope; or illuminated with an X-Cite 120Q excitation light source and viewed with a Zeiss SteREO Discovery V8 microscope equipped with a ds-Red filter.Adult flies were anesthetized on a COD. melanogaster, D. sechellia, D. virilis, and A. coluzzii animals were immobilized with clear nail polish and imaged on a Zeiss SteREO Discovery V8 microscope. Images were acquired with a smartphone camera attached to the microscope ocular and processed in Photoshop (CS6) to remove background.Whole adults were anesthetized on ice before imaging. Whole larvae, pupae, or freshly dissected adult heads were affixed to slides with clear nail polish before imaging. All animals were imaged on an Olympus SZX7 microscope equipped with GFP and RFP filters. Animals were illuminated with an X-Cite Series 120Q light source. Images were acquired using a QImaging QIClick Cooled digital CCD camera and Q-Capture Pro 7 software. Multiple images were taken at different focal planes and then merged in Photoshop (CS6). Gain was adjusted in Fiji. Images appear in the following figures/panels: All flies were used at 3–11 days old. Apart from the cryosection protocols and portions of the antennal whole-mount protocol, all immunostaining steps were done on a nutator. All steps involving or following the addition of fluorescently conjugated secondary antibodies were done in the dark.Brain and VNC staining was performed as in 2Orco mutant palps, 3-day-old flies were used to check for Orco expression before neurons degenerate . Primary antibodies used: rabbit anti-Ir25a , rabbit anti-Orco , and rat anti-elav . Secondary antibodies used: Cy3 goat anti-rabbit , Alexa 647 goat anti-rabbit , and Alexa 647 goat anti-rat . For whole-mount staining of generate .2, 2 mM MgCl2, 25 mM HEPES], 100 µL DMSO) on ice, then warmed on a 37°C heat block for 10 min. Heads were incubated in CCD buffer at 37°C while rotating for 1 hr 20 min. Antennae were subsequently dissected off heads into fixative solution (4% PFA in PBT). All subsequent steps were done without rotation to prevent antennae from sticking to the walls or lids of the tubes. The antennae were fixed at RT for 40 min, then washed with PBT three times at RT, at least 15 min each time, and blocked in PBT plus 5% NGS for at least 1 hr at RT. The antennae were incubated in primary antibodies in blocking solution at 4°C for 4 days, washed three times for 15 min each at RT, and incubated in a secondary antibody solution at 4°C for 3 days. The antennae were then washed three times for 15 min each time at RT and mounted in SlowFade Gold. Primary antibody: rabbit anti-Orco . Secondary antibody: Cy3 goat anti-rabbit . The endogenous GFP signal was visualized.The protocol for whole-mount staining of antennae was adapted from The cryosection protocol was adapted from For sacculus staining, 7- to 10-day-old flies were placed in an alignment collar. Their heads were encased in OCT (Tissue-Plus Fisher) in a silicone mold, frozen on dry ice, and snapped off. The head blocks were stored in centrifuge tubes at –80°C. A Leica cryostat was used to collect 20 µm sections of antennae. Immunohistochemical staining was carried out by fixing tissue in 4% paraformaldehyde for 10 min, followed by three 5 min washes in 1× PBS. The tissue was washed in 1× PBS containing 0.2% Triton-X (PBST) for 30 min to permeabilize the cuticle. Lastly, the tissue was washed in PBST containing 1% bovine serum albumin (BSA) to block nonspecific antibody binding. Primary antibody solution was made in PBST + 1% BSA, 200 µL was pipetted onto each slide under bridged coverslips, and slides were placed at 4°C overnight to incubate. The following day, the primary antibody was removed, and the slides were washed three times for 10 min each in PBST. Secondary antibody solution was made in PBST + 1% BSA, 200 µL was pipetted onto each slide under bridged coverslips, and left at RT in a dark box to incubate for 2 hr. After the 2 hr incubation, the slides were washed three times for 5 min each in PBST. After the last wash, the slides were allowed to dry in the dark staining box for ~30 min before being mounted in Vectashield, coverslipped, and stored at 4°C. Primary antibodies: rabbit anti-Ir25a , guinea pig anti-Ir8a , and rabbit anti-Ir64a . Secondary antibodies: Jackson Immuno Cy3 conjugated AffiniPure 568 goat anti-rabbit  and Alexa Fluor 568 goat anti-guinea pig .Cryosectioning for antennal in situs was performed as described above. The HCR protocol was adapted from Molecular Instruments HCR v3.0 protocol for fresh frozen or fixed frozen tissue sections . Slides Drosophila species tested in this paper was adapted from a combination of D. melanogaster whole-mount embryo protocol from Molecular Instruments : Drosophila simulans , Drosophila erecta , Drosophila ananassae , Drosophila pseudoobscura , Drosophila mojavensis , and Drosophila virilis . While we could detect a clean signal for the D. mel. Orco probe in these other fly species, the high background and poor signal-to-noise ratio for D. mel. Ir25a prevented co-localization analyses for all species but D. sechellia.The whole-mount palp in situ protocol for all truments . All steAnopheles in situs were performed essentially as described above, with the following modifications: olfactory appendages were dissected from 5- to 6-day-old female mosquitoes and incubated in CCD buffer for either 20 min (antennae) or 1.5 hr  at 37°C while rotating. The tissue was then pre-fixed for 24 hr at 4°C. After the PBS-Tween and methanol/DMSO washes, the tissue was incubated overnight at –20°C in absolute methanol. The next day, the tissue was rehydrated in a graded methanol/PBS-Tween series. Subsequent steps follow the Drosophila whole-mount palp protocol. Probe concentration was 8 pmol in hybridization buffer (two night incubation), and hairpin concentration was 18 pmol in amplification buffer (one night incubation). The tissue was rinsed three times in SlowFade Diamond before being mounted.Orco and the B4 amplifier conjugated to Alexa 488 for Ir25a. For single-color in situs , we used the B2 amplifier conjugated to Alexa 647.For two-color in situs, we used the Molecular Instruments B2 amplifier conjugated to Alexa 647 for Brains, VNCs, antennae, maxillary palps, and antennal cryosections were imaged on a Zeiss LSM 700 confocal microscope equipped with Fluar 10×/0.50 air M27, LCI Plan-Neofluar 25×/0.8 water Korr DIC M27, Plan-Apochromat 40×/1.3 Oil DIC M27, and C-Apochromat 63×/1.2 water Korr M27 objectives. Images were acquired at either 512 × 512 or 1024 × 1024-pixel resolution with 0.43, 0.58, 2.37, or 6.54 µm z-steps. For illustration purposes, confocal images were processed in Fiji/ImageJ to collapse Z-stacks into a single image using maximum intensity projection. Where noted, single slices or partial z projections were used as opposed to full stacks. For co-labeling experiments, Fiji was used to convert red Look-Up Tables (LUTs) to orange for a colorblind-friendly palette. Similarly, in For sacculus staining , slides Magnification used:10×: Figure 2—figure supplement 1E25×: Figure 3—figure supplement 1A and B40×: 7D and E63×: Figure 7CIr8a knock-in expression: in the ALs, we found that the sparse Ir8a+ expression in olfactory neurons targeting VM6m and VM6v could potentially be sexually dimorphic. Male brains generally had stronger and more frequent Ir8a+ innervation in these two glomeruli compared to female brains, as shown in Note regarding 2-2H2O in 1 L H2O). Extracellular activity was recorded by inserting the glass electrode into the shaft or base of the sensillum of 3- to 10-day-old flies (unless otherwise specified in the young 2Orco mutant experiments). A tungsten reference electrode was inserted into the fly eye. Signals were amplified 100× , input into a computer via a 16-bit analog-digital converter, and analyzed offline with AUTOSPIKE software . The low cutoff filter setting was 50 Hz, and the high cutoff was 5 kHz. Stimuli consisted of 1000 ms air pulses passed over odorant sources. The Δ spikes/s was calculated by counting the spikes in a 1000 ms window from ~500 ms after odorant stimuli were triggered, subtracting the spikes in a 1000 ms window prior to each stimulation. For ab3 recordings from wildtype, 2Orco mutant, and 2Ir25a mutant flies, spikes were counted in a 500 ms window from the start of the response and multiplied by 2. Then, the spikes in the 1000 ms window prior to stimulation were subtracted from this to calculate the Δ spikes/s. Stimuli used: mineral oil (Sigma CAS# 8042-47-5), EA (Sigma CAS# 141-78-6), PA (Sigma CAS# 628-63-7), benzaldehyde (Sigma CAS# 100-52-7), ethyl butyrate (Sigma CAS# 105-54-4), hexanol (Sigma CAS# 111-27-3), e2-hexenal (Sigma CAS# 6728-26-3), geranyl acetate (Sigma CAS# 105-87-3), 2-heptanone (Sigma CAS# 110-43-0), 1-octen-3-ol (Sigma CAS#3391-86-4), 2,3-butanedione (Sigma CAS#431-03-8), phenylacetaldehyde (Sigma CAS# 122-78-1), phenethylamine (Sigma CAS# 64-04-0), propionic acid (Sigma CAS# 79-09-4), 1,4-diaminobutane (Sigma CAS# 110-60-1), pyrrolidine (Sigma CAS# 123-75-1), p-cresol (Sigma CAS# 106-44-5), and methyl salicylate (Sigma CAS# 119-36-8). Odorants were dissolved in mineral oil at a concentration of 1%, and 20 µL of solution was pipetted onto filter paper in a glass Pasteur pipette. Stimuli were delivered by placing the tip of the Pasteur pipette through a hole in a plastic pipette  that carried a purified continuous air stream (8.3 mL/s) directed at the antenna or maxillary palp. A solenoid valve (Syntech) diverted delivery of a 1000 ms pulse of charcoal-filtered air (5 mL/s) to the Pasteur pipette containing the odorant dissolved on filter paper. Fresh odorant pipettes were used for no more than five odorant presentations. 2Ir25a and 2Orco mutant fly lines were outcrossed into the 1118 wildtypew genetic background for at least five generations. Full genotypes for ab3 fgSSR were Pin/CyO;Or22a-Gal4,15XUAS-IVS-mcd8GFP/TM6B (wildtype), 2;Or22a-Gal4,15XUAS-IVS-mcd8GFP/TM6BIr25a (2 mutantIr25a), and 2Or22a-Gal4/10XUAS-IVS-mcd8GFP (attp40);Orco (2 mutantOrco). These stocks were made from the following Bloomington Stocks (outcrossed to the Potter lab 1118w genetic background): BDSC# 9951, 9952, 23130, 32186, 32193, and 41737.Flies were immobilized and visualized as previously described . Basiconwildtype flies, ac2 sensilla were identified by their strong responses to 1,4-diaminobutane and 2,3-butanedione. The absence of a strong response to ammonia was used to rule out ac1 sensilla, the absence of a hexanol response was used to rule out ac3 sensilla, and the absence of a phenethylamine response was used to rule out ac4 sensilla. In Ir25a mutant flies in which amine responses were largely abolished, ac2 and ac4 sensilla were distinguished based on anatomical location, as well as the strong response of ac2 to 2,3-butanedione and the moderate response to propanal (both absent in ac4). ac1 and ac3 sensilla were excluded similarly in the mutant and wildtype flies. No more than four sensilla per fly were recorded. Each sensillum was tested with multiple odorants, with a rest time of at least 10 s between applications. The odorants used were acetic acid , ammonium hydroxide , cadaverine , hexanol , 2,3-butanedione , phenethylamine , propanal , and 1,4-diaminobutane . Odorants were diluted in water or paraffin oil. Odorant cartridges were made by placing a 13 mm antibiotic assay disc (Whatman) into a Pasteur pipette, pipetting 50 µL odorant onto the disc, and closing the end with a 1 mL plastic pipette tip. Each odorant cartridge was used a maximum of four times. The tip of the cartridge was inserted into a hole in the main airflow tube, and odorants were applied at 500 mL/min for 500 ms. Delivery was controlled via LabChart Pro v8 software (ADInstruments), which directed the opening and closing of a Lee valve (02-21-08i) linked to a ValveBank 4 controller (AutoMate Scientific). Extracellular action potentials were collected with an EXT-02F amplifier (NPI) with a custom 10X head stage. Data were acquired and AC filtered (300–1700 Hz) at 10 kHz with a PowerLab 4/35 digitizer and LabChart Pro v8 software. Spikes were summed in coeloconic recordings due to their similar sizes, and they were counted over a 500 ms window, starting at 100 ms after stimulus onset.Coeloconic SSR was performed similarly as for basiconic sensilla. Three- to five-day-old female flies were wedged in the tip of a 200 µL pipette, with the antennae and half the head exposed. A tapered glass electrode was used to stabilize the antenna against a coverslip. A BX51WI microscope (Olympus) was used to visualize the prep, which was kept under a 2000 mL/min humidified and purified air stream. A borosilicate glass electrode was filled with sensillum recording solution  and inseIr25a-T2A-QF2 was crossed to QUAS-CsChrimson #11C and double balanced to establish a stable stock . Newly eclosed flies (age <1 day old) were transferred to fly vials containing 0.4 mM all trans-retinal in fly food . Vials with flies were kept in the dark for at least 4 days before experiments. 627 nm LED light source  powered by an Arduino Uno (https://docs.arduino.cc/hardware/uno-rev3/) was used to activate CsChrimson. By setting the voltage to 2 V and the distance of the light source to 20 cm between the LED and the fly antenna, the light intensity was equivalent to 1.13 W/m2. The antenna was stimulated for 500 ms followed by 5 s of recovery period for the total recording length of 20 s (three stimulations). The identity of ab2 and ab3 sensilla were first verified with 1% EA (Sigma #270989) and 1% PA (Sigma #109584) before optogenetic experiments. Identification of ab9 sensilla was assisted by fluorescence-guided SSR (fgSSR)  recombined with 15XUAS-IVS-mCD8::GFP (BDSC #32193). The Δ spikes/s was calculated as for other basiconic SSR. For all optogenetic experiments, the control flies were of the same genotype as experimental flies but had not been fed all-trans retinal. (fgSSR)  using OrIr co-receptors across all OSNs were lower than for Orco, even for their corresponding ‘canonical’ glomeruli. To account for these differences and facilitate comparisons, we performed within-gene normalization in Dataset analyzed in this paper was published in https://flywire.ai/; navis (https://github.com/schlegelp/navis) and fafbseq (https://github.com/flyconnectome/fafbseg-py). OSNs were clustered using FAFB synapse predictions (navis). The reconstructions can be viewed at https://flywire.ai/#links/Task2021a/all.VM6 OSNs  were recdictions  for a syD. melanogaster, D. sechellia, and D. virilis Orco sequences were compared using the FlyBase (https://flybase.org/) BLAST tool . The A. coluzzii Orco sequence was downloaded from VectorBase (https://vectorbase.org/) . The pea aphid Orco sequence was acquired from the European Nucleotide Archive (https://www.ebi.ac.uk/ena/browser/view/AQS60741) (sequence reference ENA|AQS60741|AQS60741.1). Sequences were aligned using MUSCLE in MEGA11 software. This alignment was used to generate the phylogenetic tree shown in Acyrthosiphon pisum) outgroup, thought to represent one of the most evolutionarily ancient examples of functional Orco/Or complexes : within genotype, Kruskal–Wallis test with uncorrected Dunn’s to determine which odorant responses were significantly different from mineral oil or paraffin oil control; between genotype, Mann–Whitney U to compare responses of two genotypes to the same odorant . Summary tables in wildtype nor 2Ir25a mutant odor-evoked activity for given odorant was significantly different from its respective mineral oil control, nor was the difference between the genotypes statistically significant; no difference means that either wildtype or mutant or both had a significantly different odor-evoked response to the stimulus compared to mineral oil control, but the difference between the two genotypes was not statistically significant; higher response (in either wildtype or mutant) means that there was a statistically significant difference between genotypes for the given odorant. This could mean that (a) one genotype did not have a response, while the other did; (b) both genotypes had a response, and one was higher; (c) responses are different from each other, but not from their respective mineral oil controls; or (d) neural activity was inhibited by the odorant in one genotype compared to mineral oil control, and either not inhibited in the other genotype or inhibited to a lesser degree. Nonparametric tests were chosen due to small sample sizes and/or data that were not normally distributed.Statistical analyses on SSR data were done in GraphPad Prism (version 8), except for optogenetic experiments, which were analyzed in Microsoft Excel. Box plots were made using GraphPad Prism; bar graphs were made in Excel. For all analyses, significance level In  Drosophila melanogaster  are expressed widely and highly overlapping. These findings challenge a long-standing dogma in the field and suggest that different types of receptors, that is, olfactory and ionotropic receptors, can be co-expressed in the same chemosensory neuron. Moreover, optogenetics and single sensillum recordings provide evidence that IR25a co-receptor might modulate the activity of typical Orco-dependent olfactory sensory neurons. The authors also provide evidence that this co-expression is conserved by examining two other fly species.A combination of methods, including a new method for tagging genes, demonstrates that the chemosensory co-receptors of  public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:Drosophila Olfactory Neurons" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Reviewing Editor and K VijayRaghavan as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Chemoreceptor Co-Expression in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Both reviewers support publication of this study. While they are by and large satisfied with the first part of the study (description of co-expression etc.), they have indicated that the second part  could be somewhat improved.1. Please address all major points pertaining primarily but not exclusively to the functional significance of your findings.2. While additional experiments are not obligatory, we recommend the inclusion of additional controls or alternative examples (i.e. traces) as indicated in the detailed reviews below.Reviewer #1 (Recommendations for the authors):eLife. Nevertheless, the manuscript could be improved by addressing these more general points as this might help to interpret and understand in how far these findings should change our view of the olfactory system and receptor function. If this is observed in other insect species (e.g. by sequencing), this would suggest an evolutionary conserved and hence likely important feature of the chemosensory system.At this point, the observation is very interesting and should certainly be reported in – I would like to invite you to speculate a bit more about what your observation might mean and in how far it should change how we think about chemosensation.– I do appreciate that cross-species analysis is challenging, but it would make your finding a lot more impactful.– It would be great to find a way to test the importance of this co-expression in a behavioral paradigm (e.g. CO2 avoidance vs. attraction).Reviewer #2 (Recommendations for the authors):Line 47: please add ".Figure 4D: the red rectangle hides the traces. It would be nicer if it is lighter in color.Figure 6 F: It would be better if the authors replace the up trace with a better trace.Figure 2—figure supplement 2A: the example trace from the wildtype looks strange. I wonder if the authors could replace it with a better trace.Figure 4—figure supplement 1B, C: Again, the red rectangles hide the traces. The author should use Ir25a-T2A-QF2>QUAS-CsChrimson not feed all-trans retinal as a control.Line 1603-1604: It would be better if the authors mention which odorants they used exactly to identify each of ab2 and pb1-pb3 sensilla.Key resources Table: It would be better if the authors add the catalog # of the chemicals in addition to the CAS #. Essential revisions:Both reviewers support publication of this study. While they are by and large satisfied with the first part of the study (description of co-expression etc.), they have indicated that the second part  could be somewhat improved.1. Please address all major points pertaining primarily but not exclusively to the functional significance of your findings.2. While additional experiments are not obligatory, we recommend the inclusion of additional controls or alternative examples (i.e. traces) as indicated in the detailed reviews below.Orco and Ir25a expression in the maxillary palps of Drosophila sechellia, as well as in situ analyses for the palps and antennae of the malaria mosquito Anopheles coluzzii. We find that Orco and Ir25a co-expression occur broadly in these insects, addressing a reviewer concern that chemoreceptor co-expression might have been unique to a lab strain of Drosophila melanogaster. We provide a point-by-point response to the reviewers’ comments in blue below.We thank the two reviewers for their support of our work, and for their helpful and insightful comments on the manuscript. In this revised version, we updated the manuscript text and figures to address specific concerns of the reviewers. We have also included new experiments aimed at broadening the significance of our work (as requested by Reviewer 1). We performed fluorescence in situ analyses of Reviewer #1 (Recommendations for the authors):At this point, the observation is very interesting and should certainly be reported in eLife. Nevertheless, the manuscript could be improved by addressing these more general points as this might help to interpret and understand in how far these findings should change our view of the olfactory system and receptor function. If this is observed in other insect species (e.g. by sequencing), this would suggest an evolutionary conserved and hence likely important feature of the chemosensory system.eLife. To address this reviewer’s comment regarding the broader impact of this work beyond Drosophila melanogaster, we have performed a series of new experiments shown in a new Figure . We performed fluorescence in situ hybridization experiments in the palps of Drosophila sechellia. As we found for Drosophila melanogaster, we also detect broad co-expression of Orco and Ir25a in this other fly species. To extend these results even further, we performed fluorescence in situ hybridization experiments in the antennae and palps of the malaria mosquito, Anopheles coluzzii. Similarly, we found co-expression of Orco and Ir25a in both of the mosquito olfactory organs. These findings suggest that Orco and Ir25a co-receptor expression likely occurs across many insect species and is not unique to an inbred lab strain of Drosophila melanogaster.We thank the reviewer for their strong support of this work and for its publication in The new Figure 7 corresponds to a new section of the results at revised lines 509-526:Drosophila melanogaster, we used RNA in situ hybridization to examine expression of Orco and Ir25a orthologues in the fly Drosophila sechellia and in the mosquito Anopheles coluzzii . Drosophila melanogaster and Drosophila sechellia diverged approximately 5 million years ago , while the Drosophila and Anopheles lineages diverged nearly 260 million years ago  . Because co-receptor sequences are highly conserved, we could use our D. mel. Orco and Ir25a in situ probes  to examine the expression of these genes in the maxillary palps of Drosophila sechellia. We found widespread co-expression of Orco and Ir25a , consistent with our findings in Drosophila melanogaster . For Anopheles coluzzii mosquitoes, we designed Anopheles-specific Orco and Ir25a probes, and examined co-receptor co-expression in antennae  and maxillary palps . We observed broad co-expression of AcOrco and AcIr25a in the maxillary palp capitate peg sensilla , and narrower co-expression in the antennae (25% double labeled). Co-expression results for all tissues examined are summarized in Figure 7F, and cell counts can be found in Table 5. These results suggest that Orco and Ir25a co-receptor co-expression extends to other Drosophilid species as well as mosquitoes ).”“Co-Receptor Co-Expression in Other Insect Olfactory Organs. To determine if co-receptor co-expression might exist in other insects besides – I would like to invite you to speculate a bit more about what your observation might mean and in how far it should change how we think about chemosensation.As summarized above, we have expanded the Discussion section to raise additional biological ramifications of chemosensory co-receptor expression. Future experiments will allow such speculations to be refined, and possibly identify biological consequences not considered at this time.– I do appreciate that cross-species analysis is challenging, but it would make your finding a lot more impactful.Orco and Ir25a co-expression occurs in insect species beyond Drosophila melanogaster. Our new experiments are further supported by the findings of the Vosshall lab  that present compelling evidence that co-receptor co-expression similarly occurs in the mosquito Aedes aegypti. Furthermore, recent work in Anopheles coluzzii mosquitoes from the Zwiebel lab  demonstrated co-expression of Ir76b-expressing neurons with Orco. Altogether, our data and these independent studies support co-expression of chemosensory receptors across multiple insect species.As mentioned above, we performed a set of new experiments resulting in a new figure  which demonstrates that – It would be great to find a way to test the importance of this co-expression in a behavioral paradigm (e.g. CO2 avoidance vs. attraction).Gr21a-Gal4 driving UAS-Ir25a in a homozygous -; Gr63a-Ir25a double mutant background) and establishing behavioral apparatuses and assays not currently available in the lab . In addition, we feel the primary findings of our work- the broad existence of co-receptor co-expression- would not be significantly strengthened by these additional experiments. So while we share the reviewer’s fascination to extend these findings into behavioral paradigms, we feel this would be beyond the scope of the current work.We agree that testing the behavioral implications for receptor co-expression is a fascinating and important question. Rigorously addressing this question with the type of experiments suggested by the reviewer requires generating complex genotypes :Line 47: please add ".Thank you for pointing this out. The reference has been added to the revised manuscript.Figure 4D: the red rectangle hides the traces. It would be nicer if it is lighter in color.Thank you for the suggestion. We have made the red rectangle lighter in this figure and the corresponding supplement.Figure 6 F: It would be better if the authors replace the up trace with a better trace.We have made the suggested change to Figure 6F.Figure 2—figure supplement 2A: the example trace from the wildtype looks strange. I wonder if the authors could replace it with a better trace.We have replaced the trace with another example in Figure 2—figure supplement 2A.Figure 4—figure supplement 1B, C: Again, the red rectangles hide the traces. The author should use Ir25a-T2A-QF2>QUAS-CsChrimson not feed all-trans retinal as a control.We have lightened the color of the rectangle. That is indeed the control genotype that we used, but we have made that more explicit in the figure legend and text for clarity.Line 1603-1604: It would be better if the authors mention which odorants they used exactly to identify each of ab2 and pb1-pb3 sensilla.For optogenetic ab2 experiments, the odorants are listed (lines 1796-1798). These same odorants were also used for non-optogenetic SSR experiments in ab2 (this information has been added in lines 1714-1716). Briefly, we distinguished the ‘large’ antennal basiconic sensilla in the following way:– Ab1 can be ruled out due to having four spiking neurons– Ab2 and ab3 can be distinguished using ethyl acetate (EA) and pentyl acetate (PA)– Ab2 has high EA and low PA responses– Ab3 has low EA and high PA responsesSimilarly, we used EA and PA responses to distinguish the different palp basiconic sensilla (information added in lines 1709-1716):– Pb1A has high EA and high PA responses– Pb2A has low EA and low PA responses– Pb3A has no EA and high PA responsesThis is now more clearly describe in the modified methods on revised lines 1708-1716:“Basiconic Single Sensillum RecordingsFlies were immobilized and visualized as previously described . Basiconic sensilla were identified either using fluorescent-guided SSR (for ab3 sensilla) or using the strength of the A and B neuron responses to the reference odorants 1% ethyl acetate (EA) (Σ #270989) and 1% pentyl acetate (PA) (Σ #109584) (for ab2 and pb1-3 sensilla) . For example, pb1A has strong responses to both odorants, while pb3A does not respond to EA. Similarly, ab2 sensilla were distinguished from ab3 based on the A neuron responses: ab2A responds strongly to EA and weakly to PA, while ab3A neurons have the reverse response (weak EA and strong PA response).”Key resources Table: It would be better if the authors add the catalog # of the chemicals in addition to the CAS #.Thank you for the suggestion. This information has been added to the Key Resources Table.


N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA  Saponaria vaccaria L., also known as cow cockle or prairie carnation, is an annual herbaceous plant belonging to the carnation family (Caryophyllaceae). The flowering plant is a single species in its genus Vaccaria and was originally widespread in Eurasia, but nowadays is also found in North and South America, South Africa, and Australia [Saponaria vaccaria L. exist in the literature, for example Vaccaria pyramidata Medik., Gypsophila vaccaria (L.) Sm, and Vaccaria hispanica (Mill.) Rauschert. For more than 1000 years, Wang-Bu-Liu-Xing (Vaccariae semen in Chinese) have been used in traditional Chinese medicine to treat dysmenorrhea, amenorrhea, and lactation failure [Saponaria vaccaria L. contain triterpenoid saponins including gypsogenin bisdesmosides, cyclic peptides, flavonoids, and polysaccharides [Vaccaria pyramidata Medik. for the first time [ustralia . Many dicharides ,3,4. In rst time .N-glycosylase activity (EC 3.2.2.22). It was first shown on rat ribosomes that RIPs cleave off a specific adenine residue (A4324) from a conserved GAGA motif in the alpha-sarcin loop of the 28S rRNA [RIPs are distributed in over more than 100 species . However28S rRNA . As a coRicinus communis L. are composed of an enzymatically active A-chain and a B-chain with lectin-like properties which promotes the binding to galactose molecules on the cell surface [Dianthus caryophyllus L. and saporin-S6 from Saponaria officinalis L. [A distinction is made between type I and type II RIPs. Type II RIPs such as ricin from  surface ,10. That surface ,12. Type surface . Lacking surface . Only a  surface . Very prnalis L. ,16.The primary function of RIPs in the plant is not yet completely clear . DiffereRIPs have increasingly become a focus of research due to their very efficient protein synthesis inhibitory activity and the resulting high cytotoxicity. Currently, research is being conducted primarily with regard to their promising application in targeted anti-tumor therapy as conjugates with target-specific antibodies, so-called immunotoxins. More than 450 immunotoxins constructed with RIPs are published in the literature . Most ofSaponaria vaccaria L. (Vaccaria pyramidata Medik.) with a size of approximately 28 kDa and an isoelectric point (pI) of 9.5 and could identify its 30 N-terminal amino acids [Saponaria vaccaria L. when compared to other RIPs on different cell lines [Saponaria vaccaria L. appear to be promising candidates for use in targeted tumor therapy and crop protection. We now aimed to isolate the type I RIP from Saponaria vaccaria L., to determine its precise molecular mass and its complete amino acid sequence, and to characterize its thermal stability and enzymatic activity. Additionally, we attempted to investigate the distribution of type I RIPs across differently processed seed material, as well as identify the main location of type I RIPs within the seed. The main location of type I RIPs within the seed has never been characterized before.In 1995, the group of Fiorenzo Stirpe succeeded in isolating a type I RIP from no acids . The fulSaponaria vaccaria L. were available for protein purification. A crude extract with complex protein composition was obtained by aqueous extraction. In SDS-PAGE the crude extract showed promising protein bands in the mass range of 20–35 kDa typical for RIPs  we consistently found a second form with lower signal intensity (roughly one third) with a mass of 2872.4 (∆m = 30 Da)  see . In the  Da) see . The pro Da) see . The sap Da) see . The subAs shown in The sequence of sapovaccarin-S1 determined here is highly similar to other well-characterized type I RIP sequences with 83% sequence identity to gypsophilin-S and 79% sequence identity to each saporin-S6 and dianthin 30, as shown in To predict the protein structure of sapovaccarin-S1 a homology model was built B. The hiN-glycosylase activity on rabbit-reticulocyte lysate and purified rat liver ribosomes for the RIP isolated from V. pyramidata [N-glycosylase activity of isolated RIPs from S. vaccaria can also be extended to an A30-oligonucleotide substrate (A30). Weng developed an assay, which allows to quantify the enzyme activity of RIPs by measuring the adenine release from an A30-oligonucleotide substrate and the subsequent detection at 260 nm on a thin-layer chromatography (TLC) plate [N-glycosylase activity of the isolated sapovaccarin-S1 and -S2 and—for comparative purposes—the isolated gypsophilin-S and recombinant His-dianthin were analyzed with the adenine-releasing assay (Due to their cytotoxic effects type I and type II RIPs have now been studied for over 40 years for their promising use in anti-cancer therapy . The cytramidata . The N-gC) plate . N-glycong assay A. The adng assay A.N-glycosylase activity of sapovaccarin-S1 and -S2 in cells, we used label-free live-cell microscopy to investigate the cytotoxicity of sapovaccarin-S1 and -S2 in Huh-7 cells. The confluence analysis of the raw data was performed by the analysis algorithms of the software package CytoSMART. No effect on cell viability could be observed for the different concentrations shown in t-test, p ≤ 0.05). For 100 nM, 10 nM, and 1 nM the significant reduction in confluence occurred after 46 h, 45 h, and 47 h, respectively . At the end of the sapovaccarin-S1 and -S2 incubation time, a concentration-dependent cytotoxicity of sapovaccarin-S1 and -S2 was observable. A concentration of 1000 nM had the strongest cytotoxic effect compared to the others .In order to confirm the previously proven in vitro Protein thermal stability plays a key role in the development of new anti-cancer drugs, for both science and pharmaceutical industrial processes. The thermostability of sapovaccarin-S1 and -S2 was analyzed by nano-differential scanning calorimetry (DSC). In addition, DSC profiles of gypsophilin-S, which has been isolated recently by our group, and recombinantly expressed His-dianthin were recorded as a reference. The DSC profiles and the transition midpoint temperatures (Tm) are presented in N-glycosylase activity. The whole seeds and all eight seed fractions exerted N-glycosylase activity for all three ammonium sulfate fractions. With increasing ammonium sulfate concentration, enzyme activity increased—the highest enzyme activities were consistently found in the 90%-ammonium sulfate fractions. The next aim was to quantify the adenine release activity of the differently processed seed material. For this purpose, the 90%-ammonium sulfate fractions of the whole seeds, the fractionated seed material, the embryo-enriched and the perisperm-enriched seed fraction were investigated. The adenine release correlated with the total protein amount. According to the results shown in Saponaria vaccaria L.Beside the whole seeds, the Canadian Carnation Biocompany provided differently processed seed material, which allowed us to study the exact distribution of sapovaccarin-S1 and -S2 in the processed seed material see . The thrSaponaria vaccaria L. They were classified as type I RIPs in the course of this study, including their full amino acid sequence. Furthermore, we report for the first time that RIPs in Saponaria vaccaria L. are mainly located in the perisperm of the seeds. It should be noted here that the localization of RIPs was determined by evaluating the N-glycosylase activity of extracts from perisperm- and endosperm-enriched seed fractions. The localization in the perisperm might also indicate that sapovaccarin-S1 and -S2 could serve for nitrogen storage in Saponaria vaccaria L. This observation might lend some support to the hypothesis that RIPs in addition to having a defense function may also function as storage proteins in some plants. Both RIPs were isolated from the seeds by aqueous extraction, ammonium sulfate precipitation, and cation exchange chromatography. The exact amino acid sequence as well the molecular masses of sapovaccarin-S1 and -S2 were determined by PCR and mass spectrometry. Both isoforms differ only in the substitution of one amino acid at position 110 (Ala110 in sapovaccarin-S1 substituted by Thr110 in sapovaccarin-S2). Their intact masses lie within the characteristic mass range of type I RIPs [Here we report the isolation and characterization of sapovaccarin-S1 and -S2, two protein isoforms from e I RIPs . The ratN-glycosylase activity, in the future this issue might be clarified by producing the individual isoforms recombinantly by site-directed mutagenesis and determining their activity; however, this would fall outside the scope of the present study.The occurrence of RIP isoforms within the carnation family was already well described in the literature ,16,38. TType I RIPs are mostly encoded by intron-less genes . TherefoSaponaria vaccaria L. were first mentioned by Bolognesi et al. in 1995 [N-glycosidase activity. These results are essentially in agreement with ours. With the applied isolation and mass determination methods, Bolognesi et al. were not able to differentiate the two isoforms. Their assumption that the isolated protein fraction was composed of one protein, underlines the importance of accurate mass spectrometry methods for future research.RIPs from Gelonium multiflorum, were analyzed by infrared spectroscopy and two-dimensional correlation spectroscopy at neutral pH  in two different studies [N-glycosylase activity demonstrated in this study show, that sapovaccarin-S1 and -S2 seem to exhibit potential for toxin moieties in immunotoxins.Thermostability of sapovaccarin-S1 and -S2 in comparison to gypsophilin-S and His-dianthin was studied by DSC. The Tm of sapovaccarin-S1 and -S2, gypsophilin-S and His-dianthin were determined as 69 °C, 65 °C and 66 °C, respectively. Thus, the isolated mixture of sapovaccarin-S1 and -S2 showed the highest thermal stability among proteins studied. Little data on the thermostability of RIPs has been published. Saporin-S6 and gelonin, a type I RIP from  studies ,41. Tm o studies ,41. ThesSaponaria vaccaria L. (Caryophyllaceae). In addition to whole dried seeds, eight differently processed seed fractions were available: six sieve analysis fractions, an embryo-enriched fraction (EEF) and a perisperm-enriched fraction (PSF). The six sieve analysis fractions were obtained by collecting the particles which were stopped by the sieves  and the particles which have passed mesh size 200 (<200). The embryo-enriched fraction (EEF) and the perisperm-enriched fractions (PSF), which is the starch and hull fraction of the seed which remains after the embryo is removed, are achieved by separating the embryo from the rest of the seed using an impact and a roller mill and sieving.The Canadian Carnation BioProducts Company, Saskatoon, S7H 3R2, Canada provided the seeds of g for 20 min. The resulting supernatant is referred to as ‘crude extract’ and was purified by the following steps: Proteins were separated by fractionated ammonium sulfate precipitation at saturations of 30%, 60% and 90% ammonium sulfate. The precipitated proteins were resuspended in PBS and analyzed by SDS-PAGE (12.5%). The protein fractions were also tested for their N-glycosylase activity by measuring the released adenine by TLC densitometry at 260 nm [The whole seeds, sieving fraction 40, EEF and PSF had to be ground using an electric mill . The raw material of the remaining seed fractions was fine enough from the beginning. Each seed fraction was defatted twice with n-hexane (10 mL/g seed) at 4 °C for 30 min and dried at room temperature. The defatted seed fractions were extracted in PBS pH 7.4 (8 mL/g seed) by gentle stirring at 4 °C over 24 h and thereafter centrifuged at 8000× t 260 nm . The 90%Dianthus caryophyllus L. was recombinantly expressed in E. coli NiCo21(DE3) , purified by Ni-nitrilotriacetic acid affinity chromatography and analyzed by SDS-PAGE as described elsewhere [Gypsophila elegans M.Bieb. using ammonium sulfate precipitation and subsequent ion exchange chromatography. The isolation is described in detail elsewhere [N-terminally His-tagged dianthin 30 (His-dianthin) from the plant lsewhere . Gypsophlsewhere .®Nanoquant, Carl Roth GmbH, Karlsruhe, Germany).12.5% SDS-polyacrylamide gels were used for SDS-PAGE using the Lämmli method. Protein bands were stained with Coomassie Brillant Blue G250 as described elsewhere . ProteinTM laser. Data were analyzed using the software FlexAnalysis provided with the instrument. Samples were applied by the dried-droplet technique. Peptides were generated by trypsin, LysC, chymotrypsin and AspN in-gel digestion following a protocol described elsewhere [m/z range of 600–4000. α-cyano-4-hydroxycinnamic acid was used as matrix. Selected tryptic peptides got analyzed by tandem MS using the LIFT mode [Protein and peptide sequences were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS). All MALDI-TOF-MS measurements were performed with an Ultraflex-II TOF/TOF instrument  equipped with a 200 Hz solid-state Smart beamlsewhere . The masIFT mode . N-termiFor high resolution intact protein mass analysis by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) the isolated protein sample was analyzed using the Ultimate 3000 liquid chromatography system connected to a Q Exactive HF mass spectrometer via the ion max source with HESI-II probe . The following MS source parameters were used: spray voltage 3.6 kV, capillary temperature 320 °C, sheath gas 10, auxiliary gas 4, S-lens RF level 60, intact protein mode on. For the analysis 7 µL of a 10 µM protein solution were desalted and concentrated by injection on a reversed-phase cartridge  at 60 °C using buffer A  at a constant flow rate of 22 µL/min for 3 min. This was followed by a short linear gradient of 5–95% buffer B  within 10 min followed by washing and re-equilibration. Full MS spectra were acquired using the following parameters: mass range m/z 600–2500, resolution 120,000, AGC target 3 × 106, µscans 5, maximum injection time 200 ms. MS raw data were analyzed using BioPharma Finder . First, an averaged spectrum over the chromatographic peak was generated followed by spectral deconvolution using a relative abundance threshold of 20% and the function ‘consider overlaps’ turned off.https://web.expasy.org/translate/, accessed on 17 September 2021) was used to translate the DNA sequence to a protein sequence.Peptide mass fingerprinting and subsequent MS/MS-analysis enabled the identification of first sections of the amino acid sequence of sapovaccarin-S1 and -S2. In order to complete the identified peptides to a full sequence, two PCRs were conducted. Primer pair A was designed based on the peptide mass fingerprint results and primer pair B was derived from the results of the first PCR round and the N-terminus determined by Bolognesi et al. . The temHomology modeling was performed with MOE . The high resolution (1.4 Å) crystal structure of dianthin 30 with PDB ID 1RL0 served as a template). The target sequence and dianthin 30 exhibit 79% sequence identity and 95% sequence similarity. The target sequence and the sequence of dianthin 30 were aligned and checked for correct alignment.N-glycosylase activity of different samples was determined by using the adenine-releasing assay as described elsewhere [N-glycosylase buffer . Deviating from the publication the mixtures were incubated at 37 °C over night. Samples (each 10 µL) were applied to a TLC 0.25 mm pre-coated silica gel 60 glass plate with fluorescent indicator UV254  and developed by acetonitrile/water/ammonia (32%), (18:1.6:0.6). In addition, for quantification purposes different adenine amounts  were applied on the plate. Released adenine was determined by TLC densitometry at 260 nm using the TLCs canner 4 .The lsewhere . The assTo monitor the cytotoxicity of sapovaccarin-S1 and -S2 a label-free live-cell imaging system—the CytoSMART Omni system  was used. The CytoSMART Omni system is an automated brightfield microscope, scanning the complete well surface that can be placed in the incubator. The cytotoxicity studies were performed with Huh-7 cells, a hepatocyte carcinoma cell line. The Huh-7 cell line was obtained from Dr. Mirko Pinotti, University of Ferrara. Then, 8000 cells/well were seeded in 96-well plates, each well containing 150 µL Minimum Essential Medium supplemented with 10% FBS, the plate was placed on the CytoSMART Omni device. Cells were cultured at 37 °C and 5% carbon dioxide. After 24 h sapovaccarin-S1 and -S2 were added (in 20 µL PBS each 3 wells) ranging from 0.1 to 1000 nM . Control cells were only treated with PBS. Image analysis and confluence calculation was performed using the CytoSMART image analysis software package.The thermal stability of protein samples was investigated by differential scanning calorimetry (DSC). A NanoDSC  with capillary cells of 0.3 mL volume was used to carry out the calorimetric measurements. Successive heating and cooling buffer-buffer scans using PBS were repeated three times at a scanning rate of 1 °C/min and over a temperature range of 10–100 °C. During the measurements a total pressure of 3.0 atm was applied to the reference and the sample cell. Protein samples were prepared in PBS with a concentration of 0.4 mg/mL. Prior to measurements buffers and samples were degassed under vacuum for 15 min. A heating scan of each sample was recorded under the same conditions as the buffer-buffer scans. DSC data was analyzed by using the NanoAnalyze software . Buffer-buffer scans got subtracted from each sample scan.The protein sequence data reported in this paper will appear in the UniProt Knowledgebase under the accession numbers Q7M1L6 and C0HM39.


To investigate whether IL-1R-associated kinase (IRAK)-M is associated withprediabetes and type 2 diabetes (T2D).In this cross-sectional study, enrolled subjects were assigned to differentgroups according to their fasting plasma glucose (FPG) values. IRAK-M andmetabolic parameters, including fasting insulin (FINS), glycosylatedhemoglobin (HbA1c), homeostasis model assessment of insulin resistance(HOMA-IR) and beta-cell function (HOMA-β), and thioredoxin-interactingprotein (TXNIP), were evaluated. The area under the receiver operatingcharacteristic curve of IRAK-M and TXNIP for prediabetes and T2D wasdetermined.IRAK-M decreased significantly with increasing FPG levels. IRAK-M wasnegatively correlated with TXNIP, FPG, FINS, HbA1c, and HOMA-IR andpositively correlated with HOMA-β. The diagnostic cutoff value of IRAK-M was3.76 ng/mL for prediabetes and 3.45 ng/mL for T2D. After stratifying byIRAK-M (<3.76 and ≥3.76 ng/mL), patients with a higher TXNIP level showeda greater risk of prediabetes or T2D in the subgroup with low IRAK-M(<3.76 ng/mL).IRAK-M is independently and positively associated with prediabetes and T2D,while TXNIP is independently and negatively associated with prediabetes andT2D. IRAK-M and TXNIP serve as diagnostic factors for prediabetes. Toll-like receptors (TLRs), the most upstream pattern-recognition receptorsin immune cells, recruit intracellular signaling molecules, including myeloiddifferentiation primary response gene 88 (MyD88), members of interleukin (IL)-1receptor-associated kinases (IRAKs), and tumor necrosis factor (TNF)receptor-associated factor 6 (TRAF-6), leading to the activation of c-Jun N-terminalkinase, p38, and nuclear factor-kappa B (NF-κB)-dependent inflammatory responses. TLRs are activated by exogenous infectious ligands and respond to endogenousautoantigens released upon cell death or injury, inducing inflammation and leadingto the development of autoimmune diseases and self-tissue damage.Type 2 diabetes (T2D) is a metabolic disease associated with insulin resistance andpancreatic islet β-cell dysfunction. The development of T2D involves the chronicactivation of inflammatory pathways.3 The stimulation of TLRstriggers downstream pathways involving modulatory molecules and induces theproduction of the inflammatory cytokines IL-1β and IL-18. IL-1β levels are elevated upon exposure to high glucose, leading to sustainedinflammation and diabetic cell damage/cell death.6 Increased serum levels of IL-18were also observed in patients with T2D. The activation of TLR2 and TLR4 was shown to have negative effects on insulin resistance. A recent study using a T2D mouse model revealed that TLR4/MyD88/NF-κBsignaling induces the production of IL-6, TNF-α, and monocyte chemoattractantprotein, leading to the heart- and liver-related complications of T2DM. Inflammatory responses induced by TLR4 activation in human islets lead toβ-cell failure. In an in vivo study, saturated fatty acids were shown toinduce β-cell dysfunction by recruiting M1 macrophages to islets via the TLR4/MyD88 pathway. Macrophage-mediated TLR signaling may alter the islet cytokine secretome andincrease immunoreactivity in islets, suggesting that TLR signaling provides a targetfor the treatment of islet inflammation in T2D.In vivo, β-cell death and its sensing via TLR2 may be an initialevent for the stimulation of antigen-presenting cells and development of autoimmune diabetes. Additional studies revealed that the expression levels of TLR2 and TLR4proteins in monocytes in the peripheral blood of patients with T2D are significantlyhigher than those in healthy people.1416TLRs have been suggested to play a role in β-cell dysfunction and glucosehomeostasis, priming the pathogenesis of T2D. Current studies suggest a negative regulatory role of IRAK-M in TLR/IL-1Rsignaling through several mechanisms1821 The expression ofthioredoxin-interacting protein (TXNIP), which activates inflammatory pathwaysduring the immune response, is also increased in patients with T2D.23 TheTLR4/MyD88-mediated NF-κB pathway may also interact with components of inflammationin a TXNIP-associated manner.IRAKs play important roles in TLR/IL-1R signaling. IRAK family members consisting ofIRAK1, IRAK2, IRAK3 (IRAK-M), and IRAK4 mediate the activation of TLR and IL-1Rsignals and have a positive or negative regulatory effect on innate immunity,adaptive immunity, and inflammation. In addition, IRAK-M was shown to be involved in obesity-induced metabolic inflammation and atherogenesis. IRAK-M inhibits IRAK-1-mediated production of proinflammatory cytokines bypreventing the phosphorylation of IRAK-1 and dissociation of phosphorylated IRAK-1from the MyD88 complex, and the deletion of IRAK-1 improves muscle insulin sensitivity. TXNIP also regulates glucose and lipid metabolism by mediating β-cell function. As such, the downstream regulators of TLR signaling IRAK-M and TXNIP aresuggested to be involved in T2D.Inflammation is an important component underlying the pathogenesis of prediabetes andT2D. IRAK-M may mediate glucose homeostasis, directly or indirectly.IRAK-M-deficient mice are more prone to the development of type 1 diabetes (T1D) andglucose intolerance.There are limited studies on the changes in IRAK-M levels in patients withprediabetes, and its relationship with related metabolic indexes, includingindicators of β-cell function and insulin resistance, is unclear. In addition,although TXNIP was suggested to be highly increased in patients with T2D, it isunclear whether TXNIP is increased in the prediabetes stages. To investigate theassociation of IRAK-M and TXNIP changes with the development of prediabetes and T2Dand assess the use of IRAK-M and TXNIP as additional indicators of prediabetes andT2D, we measured IRAK-M and TXNIP in patients with various fasting plasma glucose(FPG) levels and determined the cutoff values of IRAK-M and TXNIP for prediabetesand T2D with receiver operating characteristic curve (ROC) analysis. All patients were newly diagnosed with prediabetes or T2D, and none hadreceived diabetes medications or dietary supplement interventions.In this cross-sectional study, consecutive subjects undergoing routine physicalhealth examination in the Second Affiliated Hospital of Ningxia MedicalUniversity, China, between August 2020 and November 2020 were enrolled. Patientswere excluded if they 1) were previously diagnosed with prediabetes, T1D, orT2D; (2) had acute or chronic infections; (3) had autoimmune diseases; (4) had ahistory of cardiovascular or cerebrovascular diseases; (5) had chronic kidneydisease, liver disease, blood disease, or cancer; (6) had thyroid dysfunction;or (7) had alcohol or drug abuse. Women who were taking hormone replacementtherapy were also excluded. Included subjects were classified into four groupsaccording to their FPG values: 1) normal control (NGT), FPG < 5.6 mmol/L; 2)prediabetes A (PD-A), 5.6 mmol/L ≤ FPG <6.1 mmol/L; 3) prediabetes B (PD-B),6.1 mmol/L ≤ FPG < 7.0 mmol/L; and 4) T2D, FPG ≥ 7.0 mmol/L. The diagnosis ofprediabetes and T2D was based on the 2021 American Diabetes Association standards.This study conformed to the principles of the Declaration of Helsinki and wasapproved by the Ethics Committee of The First People’s Hospital of Yinchuan (No.2021029). Written informed consent was provided by all subjects before theyparticipated in the study.Family history of T2D, personal history of complications, and individual pastmedical history were obtained with a unified questionnaire. Baselinecharacteristics, including systolic blood pressure (SBP), diastolic bloodpressure (DBP), height, weight, waist circumference (WC), hip circumference,body mass index (BMI), and waist-to-hip ratio (WHR), were measured. Peripheralvenous blood was collected after an 8- to 12-hour fast for laboratory tests,which included FPG, total cholesterol (TC), low-density lipoprotein (LDL),high-density lipoprotein (HDL), triglycerides (TG), alanine aminotransferase(ALT), aspartate aminotransferase (AST), creatinine (Cr), and uric acid (UA).Fasting insulin (FINS) and glycosylated hemoglobin (HbA1c) were detected by anenzyme-linked immunosorbent assay (ELISA) with a fluorescence microplate reader, and the concentrations of IRAK-M and TXNIPwere measured with an ELISA kit . Homeostasis model assessment of insulin resistance (HOMA-IR)and beta-cell function (HOMA-β) were calculated as HOMA-IR = FPG × FINS/22 andHOMA-β = 20 × FINS/(FPG−3.5). The estimated glomerular filtration rate (eGFR)was calculated with the Chronic Kidney Disease Epidemiology Collaboration(CKD-EPI) equation.Based on a preliminary estimation of a ∼15% incidence of diabetes and 40%incidence of prediabetes in a local community, a power analysis suggested asample size of at least 370 participants would be necessary, with a power of 0.8and a type I error (α) of 0.05. Data are expressed as a number (percentage) ormean ± standard deviation. Comparisons among different groups were performed bythe one-way analysis of variance, and comparisons between two groups wereperformed with student’s t-test. Pearson’s correlation test and multivariatestepwise linear regression analysis were used to assess the correlation andimpact between parameters, respectively. Logistic regression analysis was usedto assess the odds ratio of IRAK-M and TXNIP in predicting prediabetes and T2D.A ROC curve was used to calculate the specificity and sensitivity of IRAK-M andTXNIP for the diagnosis of prediabetes and T2D. All data analyses were conductedusing GraphPad Prism 8.0 software  and IBMSPSS Statistics for Windows, Version 25.0 software .P < 0.05 was considered to indicate a statistically significantdifference.Four hundred thirty subjects, including 281 men and 149 women, were included inthis study and classified into the NGT (n = 119), PD-A (n = 75), PD-B (n = 123),and T2D (n = 113) groups according to their FPG. Among all included subjects,50% had hypertension, and 30% were receiving medications .The mean age, sex, and proportion of patients with hypertension were similarbetween the four groups . HoweverIn addition, SBP, DBP, BMI, WC, WHR, TG, LDL, ALT, AST, HbA1c, FPG, FINS, andHOMA-IR increased significantly with higher FPG values, whereas HOMA-βsignificantly decreased . Subsequent multiple stepwise linear regression analysis showed thatthe IRAK-M level was significantly associated with TXNIP, HbA1c, TG, HOMA-β,FINS, and UA in all subjects  according to the datashown in 33 The development of T2D and its complications is also associatedwith abnormal immune responses and chronic inflammation, and innate immunity is highly associated with T2D. The functions of innate immune cells involved in T2D are mainly mediated viaTLRs, particularly TLR2 and TLR4. TLRs recognize exogenous and endogenous antigens and produce pro-inflammatorycytokines and chemokines, resulting in chronic inflammation that may damage β-cellsand lead to T2D.36 Several molecules are involved in TLR signaling. Among them, IRAK-M mainly restricted to monocytes/macrophages is a negativeregulator of TLR signaling pathways. IARK-M is induced upon TLR stimulation and then prevents the dissociation ofIRAK-1 and IRAK-4 from MyD88 and the formation of the IRAK-1-TRAF-6 complex. A previous study with nonobese diabetic mice found that IRAK-M deficiency(IRAK-M−/−) led to the early onset and rapid progression of T1D,accompanied by more severe insulitis and elevated anti-insulin autoantibodies. In our cross-sectional study with human subjects classified according to FPG,the level of IRAK-M was significantly decreased in patients with T2D and also duringthe prediabetes stages (PD-A and PD-B groups). Therefore, we speculate that beforeT2D develops, abnormalities in TLR signaling occur in an IRAK-M-associatedmanner.In this study, we found that the plasma level of IRAK-M was significantly lower inpatients with newly diagnosed prediabetes or T2D. IRAK-M was significantlypositively correlated with HOMA-β and significantly negatively correlated with FPG,FINS, HbA1c, and HOMA-IR. These results suggest that decreased IRAK-M is associatedwith prediabetes and T2D. Moreover, the plasma level of TXNIP was significantlyhigher in patients with newly diagnosed prediabetes or T2D. TXNIP was alsonegatively correlated with IRAK-M, suggesting that increased TXNIP is associatedwith prediabetes and T2D. These results revealed a reduced trend in IRAK-M and anelevated trend in TXNIP with increasing FPG levels. Logistic regression analysisindicated that IRAK-M is an independent negative predictor of prediabetes and T2D,while TXNIP is an independent positive predictor of prediabetes and T2D. The highAUC values for IRAK-M (0.877) and TXNIP (0.872) in prediabetes and IRAK-M (0.983)and TXNIP (0.991) in T2D further suggested that IRAK-M and TXNIP are reliablemarkers of early T2D development. T2D is a metabolic disorder associated withuncontrolled hyperglycemia and chronic inflammation that can lead to islet β-cellinjuries. Under normal physiological conditions, insulin secreted by β-cells lowersblood glucose by activating insulin receptors on the surface of various cells,including adipocytes, cardiocytes, and hepatocytes, and increases glucose uptake.However, insulin resistance affects the metabolism of several cell types, resultingin endoplasmic reticulum stress, oxidative stress, lipid homeostasis dysregulation,and mitochondrial dysfunction, which further leads to the development of T2D and itscomplications. TXNIP is an activator of the NOD-, LRR- and pyrin domain-containing protein 3(NLRP3) inflammasome, mediating oxidative stress by interacting with and inhibitingthioredoxin.39 Under high glucose conditions, the gene expression ofTXNIP is upregulated in islet cells. In addition, reactiveoxygen species (ROS) are induced under hyperglycemia, subsequently triggering therelease of TXNIP from thioredoxin, activating TXNIP/NLRP3 and a series of relatedinflammatory reactions, and leading to β-cell apoptosis.4042 Recently, NADPH oxidase 5(NOX5) was identified as a downstream target of the TLR pathway, and NOX5-derivedROS may be modulated by IRAK. Furthermore, ROS activate NLRP3 by promoting TXNIP and NIMA-related kinase 7interactions with NLRP3. The ROS production induced by IRAK may contribute to NLRP3activation initiated by the activation of these kinases.45 In addition, TXNIP was shownto be highly positively correlated with NLRP3, caspase-1, and IL-1β, and TXNIP wasabnormally expressed as the FPG level increased, suggesting that TXNIP augments thebody’s inflammatory response to oxidative stress. TXNIP inhibits insulin expression and glucose uptake, and insulin resistancecan lead to elevated TXNIP levels in islet β-cells.47 Accordingly, we speculatethat IRAK-M/TLR-related diabetes may be associated with the ROS/TXNIP signalingpathway.TXNIP is another chronic inflammation-related factor that may cause islet β-cell dysfunction. Another study revealed that TLR4 knockout activated TRIF/IRF3 signaling,induced inflammation, and increased the spleen index. Studies with TLR2 knockout (Tlr2−/−) micerevealed other possible mechanisms leading to diabetic and obese phenotypes. Forexample, the bone morphogenic protein-induced binding between TLR2 and NOX1 or NOXO1was abrogated, and the high-fat diet-induced increases in FPG and insulin levels inwild-type mice were restored in Tlr2−/− mice.The pathogenesis of T2D involving TLR-mediated chronic inflammation is complex. Inaddition to IRAK-M and TXNIP suggested in the present study, other downstreammediators of TLRs (TLR2 and TLR4) may play a role. A recent study revealed the novelregulation of TLR4 in a mouse model whereby the ribosome biosynthesis protein NOC4binds to and inhibits the endocytosis of TLR4 and blocks the TRIF pathway. Thisconsequently inhibits the production of inflammatory cytokines, subsequentlyimproving local and systemic inflammation and reducing insulin resistance. Another study revealed that IRAK-M−/− nonobese diabetic mice hadearly onset and rapid progression of T1DM and impaired glucose tolerance. However, the upregulation of IRAK-M may also imply a harmful condition. In amouse model with hypoxia-inducible factor-1α overexpression in macrophages, ahigh-fat diet increased the expression of IRAK-M, leading to macrophageinfiltration, inflammation, and fibrosis in adipose tissue. Moreover, globalIrak-M deficiency was associated with reduced fibrosis andimproved glucose tolerance, suggesting an IRAK-M-dependent mechanism inobesity-related adipose tissue dysfunction.An association between IRAK-M reduction and the development of diabetes has also beensuggested in other studies. In a study that used a dendritic cell modulator for thetreatment of T1D in nonobese diabetic mice, the induction of IRAK-M was found to beaccompanied by the increased efficacy of this drug . Here, with newly enrolled subjects, we showed similar trends in SBP, DBP,BMI, WC, WHR, TG, LDL, FPG, FINS, HOMA-IR, and HOMA-β. In addition, to furtherclassify the subjects with prediabetes into the PD-A(5.6 mmol/L ≤ FPG < 6.1 mmol/L) and PD-B (6.1 mmol/L ≤ FPG < 7.0 mmol/L)groups, we examined HbA1c, Cr, ALT, and AST in this study. When comparing the PD-Agroup with the PD-B group, only HbA1c, FINS, HOMA-IR, and HOMA-β showed significantdifferences, and there were no significant differences in blood pressure, bodyweight, BMI, WC, WHR, TG, TC, HDL, LDL, ALT, AST, UA, and Cr. These resultssuggested that during the early development of T2D, only characteristics directlyrelated to the plasma glucose level were affected. The decreasing HOMA-β values withincreasing FPG levels indicate more severe β-cell dysfunction, as expected in peoplewith T2D. However, the values of ALT, AST, and UA, which are indicators of liverdysfunction and cardiovascular disease, were the highest in the PD-B group butlowest in the T2D group. The liver is one of the main organs responsible forglycogen synthesis and glucose homeostasis maintenance, and elevated levels of liverenzymes are clinical indicators of liver dysfunction. The plasma levels of liverenzymes have been shown to be positively correlated with FPG, and patients with T2Doften have liver dysfunction.5456 Our results showed that thelevels of ALT and AST were highest in the PD-B group rather than in the T2D group;therefore, liver function might have deteriorated before the FPG level reached thediagnostic threshold for T2D. Similarly, the level of UA increased as the FPG levelincreased but was highest in the PD-B group and decreased in the T2D group. UA hasbeen suggested to be a risk factor for cardiovascular disease and dynamically associated with the level of HbA1c. In addition, the UA levelmight be higher in patients with prediabetes than in those with T2D. Our results suggested that liver and cardiovascular diseases might havedeveloped during the early stages of T2D.Previously, we reported changes in the level of various metabolic parameters andseveral physical characteristics with increasing FBG levels.60 However, as suggested by theDiabetes Control and Complications Trial, using a single measurement for FPG may be misleading. Previous studies haveshown that for patients with chronic kidney disease or microangiopathy or elderlypatients, glycated albumin is a more reliable indicator of the glycemic status thanHbA1c because of the reduced survival of erythrocytes and consequent decrease in thetime available for glucose to attach to hemoglobin.62 In addition, because subjectswith impaired FPG usually have other metabolic syndromes, cardiovascular disease, orcerebrovascular disease, additional surrogate biomarkers, including galectin-3, highsensitivity cardiac troponin I, hyperhomocysteinemia, and heart-type fatty acidbinding protein, may be considered for a more comprehensive interpretation of theunderlying pathological mechanisms.6365Glucose homeostasis or diabetes is usually assessed or identified with the surrogatebiomarkers FPG and HbA1c in clinical practice.This study has several limitations. First, the unequal numbers of subjects among thefour groups may lead to heteroscedasticity during analysis. Second, thephysiological effects of IRAK-M and TXNIP are exerted intracellularly, but onlyIRAK-M and TXNIP levels outside the cells were estimated. Third, the mechanism ofT2D development is complicated; factors other than IRAK-M, TXNIP, and metabolicindicators revealed in this study may also play a role. Fourth, albuminuria valueswere not examined in the present study, and a possible effect of early kidneydysfunction on IRAK-M and TXNIP changes cannot be ruled out. Further studies arewarranted to elucidate these issues.In conclusion, our results provide evidence that IRAK-M is independently andpositively associated with prediabetes and T2D, while TXNIP is independently andnegatively associated with prediabetes and T2D. IRAK-M and TXNIP showed high AUCvalues for patients with prediabetes. Therefore, the combination of low IRAK-M andhigh TXNIP might be diagnostic for prediabetes.Click here for additional data file.Supplemental material, sj-jpg-1-imr-10.1177_03000605221111275 for Changes inplasma IRAK-M in patients with prediabetes and its relationship with relatedmetabolic indexes: a cross-sectional study by Xiaomin Xie, Guirong Bai, LiZhang, Huili Liu, Dan Qiang and Ling Li in Journal of International MedicalResearchClick here for additional data file.Supplemental material, sj-pdf-2-imr-10.1177_03000605221111275 for Changes inplasma IRAK-M in patients with prediabetes and its relationship with relatedmetabolic indexes: a cross-sectional study by Xiaomin Xie, Guirong Bai, LiZhang, Huili Liu, Dan Qiang and Ling Li in Journal of International MedicalResearch