Moreover, aurachin D possesses remarkable activities against Leishmania donovani, the causative agent of leishmaniasis. Aurachins are naturally produced by myxobacteria of the genus Stigmatella as well as by some Streptomyces and Rhodococcus strains. The recombinant production of these antibiotics turned out to be challenging due to their complex biosynthesis and their inherent toxicity. Recently, the biotechnological production of aurachin D was established in E. coli with a titer which is higher than previously reported from natural producer organisms.Aurachins are farnesylated quinolone alkaloids of bacterial origin and excellent inhibitors of the respiratory chain in pro- and eukaryotes. Therefore, they have become important tool compounds for the investigation of electron transport processes and they also serve as lead structures for the development of antibacterial and antiprotozoal drugs. Especially aurachin D proved to be a valuable starting point for structure-activity relationship studies. Aurachin D is a selective inhibitor of the cytochrome  Natural products are an essential resource for drug development. From the past until today, many bioactive natural products with anticancer, antifungal, antibacterial, and antiprotozoal effects have been used directly as medicine or have been the inspiration for lead structures in drug development ,2,3. AmoStigmatella aurantiaca  AuaF  for the N-hydroxylation of the quinolone nucleus [Aurachin biosynthesis was initially explored in ase RauF  44]. In. InS. aurantiaca ,44. MoreStreptomyces sp. NA04227 is highly similar to that in R. erythropolis JCM 6824 (N-hydroxylation of the quinolone nucleus is carried out by a cytochrome P450 monooxygenase (SauPI or SauPII) [1 and 2. Subsequently, the derivative of 2 is converted by an unidentified methyltransferase to aurachin SS (6). Another unique feature of the aurachin biosynthesis gene cluster in Streptomyces sp. NA04227 is the presence of a thioesterase gene. The putative thioesterase SauK might contribute to the chain release from the type II PKS and the following cyclization [The biosynthesis of aurachins in JCM 6824 . A singl SauPII) . Unlike lization . An over1) might become a promising drug candidate for the treatment of bacterial and protozoal infections and it already represents a valuable pharmacological tool compound [S. aurantiaca [1, the reconstruction of its biosynthesis in a heterologous host was attempted to increase the titer and facilitate the downstream processing. Initially, R. erythropolis JCM 3201 was evaluated as a host organism for recombinant aurachin production. However, no titers were reported, and the production was accompanied by growth defects, which were attributed to the potent antibiotic activity of aurachins [Aurachin D  strategy [auaA [auaA and by improving the cellular supply of farnesyl pyrophosphate through the introduction of the mevalonate pathway. Ultimately, the titer could be raised to 17.0 mg L−1 [−1) of the natural producer S. aurantiaca [The aforementioned challenges were recently overcome with the biotransformation of 2-methyl-1 E. coli . Althougnditions ,22. The nditions . To achistrategy . The BCDstrategy . The trastrategy . A libragy [auaA . The aur0 mg L−1 , which erantiaca . 1 were generated in sufficient quantities [S. aurantiaca Sg a15 [E. coli production strain was used [Derivatization is a powerful tool to improve the activity of a natural product and obtain new insights into its target site. Generally, chemical synthesis is employed for the derivatization of natural products  and, as antities . Anothera Sg a15 . In cont1 can potentially overcome the outer membrane of a Gram-negative bacterium and are only active at the target site. This may also reduce cytotoxic effects for the patient. Furthermore, nanocarriers can protect an antibiotic from enzymatic degradation and prolong its half-time [Aurachins are a remarkable class of natural products. They are already valuable tool compounds, which have helped to gain important structural and functional insights into bacterial respiratory chains ,29,30, walf-time .1 showed potent bioactivity against Leishmania donovani, the pathogen of visceral leishmaniasis [1 need to be further investigated, an impairment of the parasite’s electron transport processes seems plausible. The leishmanial respiratory chain and, in particular, NDH2 have become promising molecular targets [P. falciparum [Leishmania, including parasites with an active complex I NADH dehydrogenase [2 was reported as a potent inhibitor of NDH2 in the rodent malaria parasite Plasmodium yoelii yoelii [1 also exerts its antileishmanial effects, at least partially, via NDH2 inhibition. Recently, the ubiquinone reduction site of cytochrome b, part of the cytochrome bc1, was demonstrated to be a promiscuous drug target in T. cruzi and L. donovani [bc1 of Rhodospirillum rubrum suggested that the ubiquinone reduction site is targeted by 2 and the oxidation site by 1. However, the inhibition of the reduction site by 1 could not be excluded [1 could also affect additional target sites in L. donovani.Especially maniasis . Althoug targets . As oppolciparum , NDH2 warogenase . Considei yoelii , one migdonovani . Early sexcluded . Of cour1 [1 in E. coli. Because gram-scale heterologous production of different isoprenoids is possible in E. coli [Besides elucidating the antileishmanial mode of action of aurachins, another prerequisite for their preclinical development is to secure a sufficient supply of these antibiotics. Some progress has been achieved by successfully establishing the heterologous production of 1 . However E. coli , carbon


Vespa velutina DPPIV sequence. Moreover, two cases of sensitization to DPPIVs in wasp- and honeybee-sensitized patients are presented. (2) Methods: Proteomic analyses have been performed on the venom of the Asian hornet Vespa velutina to demonstrate the sequence of its DPPIV . A comparison performed through their alignments and analysis of the three-dimensional structure showed a region with higher similarity among Hymenoptera DPPIVs. Additionally, ImmunoCAP™ determinations (including specific inhibition experiments), as well as IgE immunoblotting, are performed to demonstrate the allergenicity of Api m 5 and Ves v 3. (3) Results and Conclusions: The data presented demonstrate that the similarities among Hymenoptera DPPIVs are most likely localized at the C-terminal region of these enzymes. In addition, a higher similarity of the Vespa/Vespula DPPIVs is shown. The clinical cases analyzed demonstrated the allergenicity of Api m 5 and Ves v 3 in the sera of the allergic patients, as well as the presence of this minor component in the preparations used in venom immunotherapy.(1) Background: Dipeptidyl Peptidases IV (DPPIVs), present in many organisms, are minor components in the venoms of Hymenoptera, where they have been identified as cross-reactive allergenic molecules. Considering that the structure of homologous DPPIVs is well characterized, we aimed to explain which regions have higher similarity among these proteins and present a comparison among them, including a new  Vespa velutina [Vespa genera, and probably in other Hymenoptera, seems related to the maturation of the lytic toxin melittin or that of mastoparan B [Dipeptidyl Peptidases IV (DPPIVs) are type II transmembrane serine proteases, also known as CD26 or adenosine deaminase binding protein . As enzyvelutina . The phyoparan B ,8.Human DPPIV has been structurally very well characterized ,2, and iApis/Vespula, apart from the hyaluronidases, due to the similarities between Api m 5 and its counterpart in Vespula spp., named Ves v 3 [Polistes dominula’s Pol d 3 [Vespa velutina, the invasive Asian hornet that has spread over several European countries [DPPIVs are considered responsible for cross-reactivity among Hymenoptera venoms ,4,5,6,7, Ves v 3 . The com Ves v 3 ,11. This Pol d 3  as is thountries ,13. In tountries ,15, is pThe following sections describe a new DPPIV sequence, the similarities among Hymenoptera DPPIVs and several structural features of these proteins, in relation with their cross-reactivity.Vespa velutina transcriptome [Vespa velutina venom preparations from insects in the north-west regions  of the prepared Spanish peninsula, as described in [Vespa velutina venom, with 69 peptides identified in the analyses. The newly confirmed Vesp v 3 sequence will be available in the UniProtKB database [Vespa DPPIV sequence described to date , for whVespula vulgaris and Apis mellifera are shown in Vespa and Vespula genera. Very high similarity values (93% sequence identity) were also found among the hyaluronidases of these two genera, as shown by Monsalve et al. [Vespula and Vespa) and Polistes is much lower, with 76.6% sequence identity [The mature sequence of Vesp v 3 and its alignment with the counterparts from e et al. . The DPPidentity .Sequence similarities between wasps and hornet DPPIVs with Api m 5 have much lower values, with 54% sequence identity, as can be observed in the overall alignment shown in Vespa/Vespula similarity to Api m 5 increases from 65% to 85% (from 47% to 73–74% if we consider sequence identity). This much higher similarity among these proteins at the C-terminal domain of the DPPIV provides a significantly increased chance of antigenic cross-reactivity among Hymenoptera DPPIVs, and this area of the DPPIV is more likely to contain common antigenic determinants. Panel c in A BLAST sequence alignment  of the VOne additional confirmation of the structural similarity among DPPIVs in this particular domain is the fact that the amino acids responsible for the enzymatic activity of this domain , are coTwo clinical cases of Hymenoptera patients with peculiar allergenicity to DPPIV, detected by its reactivity to the ImmunoCAP™ component Api m 5, are presented in this section of the manuscript.Vespula/Polistes inhibition studies. The inhibitions required a higher concentration than usual of the Vespula and Polistes extracts (up to 100 µg of the extracts were necessary) to achieve significant inhibition, even in the case of “homologous inhibitions” , and it increased further using 100 µg of the Polistes dominula extract .A 44-year-old woman with previously reported extensive local reactions after Hymenoptera stings was urgently referred for an immediate response after an additional Hymenoptera sting by an unidentified insect with the following symptoms: palmoplantar pruritus, generalized urticaria, eyelid and labial angioedema, sensation of chest tightness, and dyspnea. She was immediately treated with adrenaline, methylprednisolone, and dexchlorpheniramine, and her symptoms disappeared within 12 h. After treatment, a complete allergologic study was performed, including the “in vitro” testing presented in this manuscript, including Component Resolved Diagnosis (CRD) and CAP-inhibition studies. bitions” a,b, for hibition a, after dominula b, with sPolistes dominula extract only inhibited the Vespula-specific IgE in the solid phase up to 53%, even with the highest quantity of the Polistes dominula extract  indicated that the patient was primarily sensitized by Vespula species, and a VIT treatment with ALUTARD SQ®-Vespula spp. was started in July 2022. The evolution of the patient’s sIgE, total IgE, and IgG4 values is shown in The “reciprocal inhibitions” c,d showe extract c. On theVespula spp. extract needed to obtain a clear inhibition of the sIgE) revealed the significant relevance of minor components of the venom extract. As a matter of fact, this patient showed a very unusual sIgE value toward the Api m 5 component in ImmunoCAP™ , 0.19 for rApi m 1 (i208), 0.0 for rApi m 2 (i214), 0.01 for rApi m 3 (i215), 0.0 for Api m 4 (h3x as ALK reference), and 0.14 for rApi m 10 (i217). Considering these generally very low or negative sIgE values, we also tested additional components with ImmunoCAP with the following results: 2.54 for rApi m 5 (i216), 0.28 for Vespula spp. Ext. (i3), 0.05 for rVes v 1 (i211), and 0.28 rVes v 5 (i209); the total IgE was 15 kU/L. These results provide insights into a predominant sensitization to Api m 5 in this Apis mellifera-sensitized patient since this was the only component showing clear sIgE positivity, with a similar value to that of the whole Apis mellifera extract. Moreover, after a complete hematological study including a bone marrow biopsy, the patient was diagnosed with bone marrow mastocytosis .Serum from a patient who suffered three syncopal episodes and sphincter relaxation after suffering bee stings was analyzed using ImmunoCAP-250, and the initial “in vitro” determinations showed the following sIgE values (KAU/L): 2.75 for ®-Apis mellifera extracts were used. These data confirm the relevance in this patient of this minor component of the Apis mellifera venom. The low abundance of Api m 5 was clearly shown in the SDS-PAGE and IgE-immunoblot (Apis mellifera extract in the SDS-PAGE (lane 2), and the sIgE reactivity toward Api m 5 is weak .Sera from the patient cases presented in this manuscript have been tested in IgE-immunoblotting experiments to observe the specificity in each case . The casis serum . The strApis mellifera extract, a positive response around the 90kDa band , with Apis being more dissimilar (70% similarity). The similarity within the Vespa genera is also very high: 97% sequence identity was found between the DPPIVs of Vespa basalis [Vespa velutina.In this manuscript, we presented the complete sequence data of the mature DPPIV component present in  species ,7 and th species ,15. We cVespula/Vespa and Apis is observed  to achieve an inhibition of 66.3%  during the winter of 2022 and was stored at −20 °C until it was used in the study. After sex and female caste differentiation, based on external morphological characteristics, the venom sacs were extracted from frozen insects by pulling the stinging apparatus from the wasp abdomen with forceps. The venom sacs of 32 workers were dissected from their posterior apparatus. Eight venom sacs were then pooled, resulting in a total of four pooled samples, representing four biological replicates (named VV 1 to VV 4). The pooled samples were then eluted through a Spin-X 0.45 µm cellulose acetate centrifuge filter , and the residual tissue from each venom sac was removed via centrifugation at 14,000 rpm for 10 min. The Spin-X was then washed with 250 µL PBS  and centrifuged at 3000 rpm for 3 min. The eluted total venom protein was transferred to a new tube and stored at −20 °C. Total venom proteins were quantified using the Bradford method .The The tryptic digestion for mass spectrometry (MS) was performed with 24 µg protein concentrated in an SDS-PAGE single band ,36, follVespa + Vespa velutina + Apis mellifera + poison + toxins UniProtKB databases available online (https://www.uniprot.org/ (accessed on 2 February 2023)) was used, specifying iodoacetamide as alkylation of the cystines and digestion by trypsin. A pool was created using 3 µL of the peptides extracted from each individual sample (VV 1–4). The pool was chromatographed for a total time of 40 min and analyzed using a data-dependent acquisition (DDA) method in a positive ion mode to build the MS/MS spectral libraries, as previously described [Mass spectrometry analysis was performed using a hybrid quadrupole-TOF mass spectrometer 6600+  coupled to an Ekspert nLC425 micro-liquid chromatography (LC) system . ProteinPilot software v.5.0.1.  was used for protein and peptide identification. A customized database containing escribed ,39,40. Tescribed . The MS/Vespa basalis sequence were selected to better define and confirm the new peptide sequence.Skyline v.3.7 was used to process the DDA and DIA data files. The new DPPIV sequence was used to generate the best monoisotopic peak of each precursor ion. This peak was extracted using the software’s default settings for MS1 quantification. In addition, the best transitions for MS2 of each precursor were also selected among the DIA files. After importing the raw data into the Skyline software, the data files were queried against the protein sequence; peptide and fragment signals were manually checked to ensure correct peak assignment and peak boundaries. Those peptides with an amino acid different from the previously known The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner Vespa velutina DPPIV was obtained, as indicated in [Vespa velutina venom glands [The sequence for the cated in , from thm glands . The Clum glands  was usedm glands . Homologm glands .SDS-PAGE was performed on 4–20% acrylamide BIO-RAD’s Mini-Protean TGX Stain-Free gels. Immunoblots with patient sera were performed after transferring to nitrocellulose membranes using Trans-Turbo Blot (BIO-RAD) equipment. Serum was diluted 1/5, and specific IgE was detected using a mouse monoclonal anti-human IgE ; finallyVespula spp. venom, crude Polistes dominula venom , or Aquagen SQ®-Apis mellifera (from ALK-Abello A/S). The Aquagen preparation was specifically used for inhibition of the Api m 5 component with the indicated concentrations . In the case presented in Vespula spp. versus Vespula spp. in the solid phase and Polistes dominula versus Polistes dominula in the solid phase) and for the “reciprocal” inhibitions (Polistes dominula extract inhibiting Vespula spp. in the solid phase and Vespula spp. extract inhibiting Polites dominula in the solid phase). Patient serum and inhibitor extracts were preincubated at room temperature for 2 h prior to the assay in the ImmunoCAP-250. The extent of homologous and heterologous venom inhibition was calculated considering the decrease in sIgE value for the corresponding component in the solid phase . Homologous inhibition was usually close to 100% using 25 µg of the pure extract, while inhibition values above 70% were considered an indication of extensive cross-reactivity [sIgE reactivity levels to the different components, as well as total IgE and IgG4-specific determinations, were obtained using ImmunoCAP-250 equipment . Apart from several commercial components from Thermofisher, sIgE to Pol d 1 (purified as described in ) was alsactivity ,26,45.


We demonstrate the strength of this approach through its ability to identify 14-3-3 binding sites that do not conform to the loose consensus sequence of 14-3-3 docking phosphorylations, which we validate with 14-3-3 client proteins, including TNK1, CHEK1, MAPK7, and others. In addition, by using this approach, we identify a phosphorylation on A-kinase anchor protein-13 (AKAP13) at Ser2467 that dominantly controls its interaction with 14-3-3.14-3-3s are abundant proteins that regulate essentially all aspects of cell biology, including cell cycle, motility, metabolism, and cell death. 14-3-3s work by docking to phosphorylated Ser/Thr residues on a large network of client proteins and modulating client protein function in a variety of ways. In recent years, aided by improvements in proteomics, the discovery of 14-3-3 client proteins has far outpaced our ability to understand the biological impact of individual 14-3-3 interactions. The rate-limiting step in this process is often the identification of the individual phospho-serines/threonines that mediate 14-3-3 binding, which are difficult to distinguish from other phospho-sites by sequence alone. Furthermore, trial-and-error molecular approaches to identify these phosphorylations are costly and can take months or years to identify even a single 14-3-3 docking site phosphorylation. To help overcome this challenge, we used machine learning to analyze predictive features of 14-3-3 binding sites. We found that accounting for intrinsic protein disorder and the unbiased mass spectrometry identification rate of a given phosphorylation significantly improves the identification of 14-3-3 docking site phosphorylations across the proteome. We incorporated these features, coupled with consensus sequence prediction, into a publicly available web app, called “ Although the number of isoforms differs among organisms, 14-3-3s are structurally and functionally conserved from plants to animals, reflecting their role in fundamental cellular processes, including cell cycle, apoptosis, and metabolism.25 the control of cell death by 14-3-3 binding to the BH3-only protein BAD,9 the control of transcription factor access to DNA,29 and the regulation of various metabolic enzymes, kinases, and structural proteins. (reviewed in14) 14-3-3s exert their effect by docking to one or two phosphorylated Serines or Threonines on the client protein.33 These phosphorylations often occur in regions of high intrinsic disorder,39 perhaps reflecting the flexibility needed for a client protein to fit within the 14-3-3 phospho-binding groove. Because these client protein phosphorylations (often in pairs) can be added by different kinases, 14-3-3 has the ability to integrate multiple signaling inputs to produce one biological output, thereby acting as a biological logic gate.40 In turn, the effect of 14-3-3 binding on the client protein can vary. 14-3-3s can sequester proteins in different cellular locations , contort the shape of the protein into active or inactive conformations, or affect the interaction of the client protein with other proteins.40Many textbook mechanisms of molecular biology have been discovered from the perspective of understanding how 14-3-3 docks to and controls client protein activity. These mechanisms include the control of cell cycle progression via 14-3-3-mediated regulation of the CDK1-regulating phosphatase CDC25,50 our understanding of how 14-3-3 regulates most of these proteins lags far behind, leaving many undoubtedly important biological mechanisms undiscovered. In our group’s experience, the most critical bottleneck of this workflow is the identification of the phosphorylation sites that mediate 14-3-3 binding. This process is low throughput and resource-consuming, generally entailing the testing of numerous sites individually and in combination through mutagenesis and coimmunoprecipitation—a process that can take many months to years for a single client protein. Yet, without identifying these phospho-sites, it is essentially impossible to gain reliable insight into the effect of 14-3-3 on the client protein.Although modern mass spectrometry has helped identify hundreds of 14-3-3 client proteins,in vitro,31 it exists in less than half of experimentally validated 14-3-3 docking sites.51 In addition, the −3 Arginine may, at least in part, reflect the consensus sequence of the many AGC family kinases that phosphorylate 14-3-3 docking sites rather than a strict requirement for 14-3-3 docking, as several other amino acids  are relatively common at the −3 position.51 Furthermore, client proteins often have numerous “red-herring” phosphorylation sites within sequences that resemble the 14-3-3 consensus docking site but have no measurable impact on 14-3-3 binding when tested in the lab. Nevertheless, mutational studies suggest that 14-3-3s do not interact promiscuously with off-target phosphorylated Serines and Threonines. Thus, protein features beyond the 6-amino acid consensus sequence must contribute to creating an optimal 14-3-3 docking site.52 This idea is supported by recent structures of 14-3-3 binding to different conformations of BRAF, which highlight a variety of contacts between 14-3-3 and BRAF outside of the 6-amino acid consensus phospho-site sequence.53Historically, 14-3-3 docking sites have been described as following the general consensus sequence of RXXpS/TXP (mode I) or RXF/YXpSXP (mode II). However, as the list of 14-3-3 client proteins has grown, we have seen many 14-3-3 docking sites differ from this sequence. For example, although the +2 proline enhances 14-3-3 binding to phospho-peptides In this study, we analyze experimentally validated 14-3-3 docking site phosphorylations to identify protein features that distinguish docking phosphorylations from non-docking phosphorylations. We also mine publicly available phospho-proteome data to gain insight into the unbiased observation frequency of docking site phosphorylations compared to all other phosphorylations across the proteome. We find that the unbiased mass spectrometry observation frequency of a given phosphorylation can reliably predict many 14-3-3 docking site phosphorylations. In addition, the degree of predicted intrinsic disorder around the phosphorylation site can further improve these predictions. By combining these features with consensus site prediction, we improve our ability to predict 14-3-3 docking site phosphorylations that match poorly with the consensus sequence. Lastly, we incorporate all these features into a machine learning-based web-app, termed “14-3-3 site-finder”, which we use to identify a phosphorylation on the guanine nucleotide exchange factor AKAP13 that controls 14-3-3 binding.https://pearl.biosci.gatech.edu). Sites were classified as either matching a “14-3-3 interacting” site or “other” and statistically evaluated using the test for unequal variances in JMP 16.1 (SAS Inc.) and the Welch’s T test that is optimal for such dataset comparisons.Phosphorylation site features aggregated at the protein and protein family level for each 14-3-3 docking site were compared to 202,850 unique S/T phosphorylation sites contained within the SAPH-ire dataset (PMID 35696084), derived from phosphosite data curated from HPRD 9.0 (PMID: 18988627), PhosphoELM (PMID 21062810), PhosphoSitePlus (PMID 30445427), SySPTM (PMID 24705204), and dbPTM (PMID 26578568). Of the 170 unique 14-3-3 sites available, 115 were also found in and could be compared with the SAPH-ire dataset from 2019 (accessible via https://github.com/lynsiewarr/14-3-3-Site-Finder.The training data set was a combination of previously identified 14-3-3 docking sites and extended from current literature. We webscraped the necessary data from IUPred2A and 14-3-3Pred. We generated a database of PTM observation frequencies from available data on PhosphoSitePlus through 2021. The tool was implemented using R with the “randomForest” library. All code is deposited here: 2.HEK293T cells were purchased from ATCC. Cells were cultured in DMEM supplemented with 10% FBS and at 37 °C with 5% COFLAG-TNK1 expression plasmid was purchased from GenScript. FLAG-AKAP13 was purchased from Addgene. Mutagenesis of constructs was done using Q5 Site-Directed Mutagenesis Kit (NEB), following the manufacturer’s protocol. Cells were plated the day prior to transfection. The transfection complex was prepared in a 1:4 DNA/PEI MAX ratio in DMEM, mixed and then incubated at room temperature for 20 minutes. After incubation, complexes were added to the cells and returned to the incubator. Media was aspirated and replaced after 6–8 hours.™ Agarose (Chromotek) for 1 hour at 4 °C with rotation. The resins were washed with PBS three times. The coimmunoprecipitated proteins were eluted with SDS sample buffer and boiled at 95 °C for 5 mins. Lysates were resolved by SDS-PAGE and transferred to nitrocellulose membrane using iBlot2 Western Blotting System. Membranes were blocked with either 5% non-fat dry milk in PBS, intercept blocking buffer (Li-cor), or 5% phosphoblocker (Cell Biolabs) in PBST for 1 hour at room temperature. Primary antibodies against proteins of interests were diluted 1:1000 in blocking buffer and incubated with blot overnight at 4 °C. Proteins were visualized and quantified using infrared fluorescent secondary antibodies IRDye® 800CW Goat anti-Rabbit , IRDye® 680RD Goat anti-Mouse  and Li-Cor Image Studio 5.0 software.For TNK1 and AKAP13 immunoprecipitations, transfected HEK293T cells were washed and harvested in cold PBS. Cell pellets were lysed in either co-IP lysis buffer  supplemented with protease and phosphatase inhibitors and rotated at 4 °C for 15 mins. Lysates were then homogenized by passaging through a 25G needle and centrifuged at 21000xg for 10 mins to clarify. Clarified lysates were collected for immunoprecipitation and western blot. For lysates were incubated with anti-DYKDDDDK G1 Affinity Resin (GenScript) or DYKDDDDK Fab-TrapPRISM GraphPad 9.0 was used for statistical analysis of data and generation of figure graphs.49 and compared these with 202,850 unique S/T phosphorylations curated in 2019. These sites were organized further using SAPH-ire, a machine learning tool for predicting functional PTM sites that also provides a rich resource for PTM site features at both the protein and protein family level.55 From this we determined which protein and familial features were highly enriched in 14-3-3 phosphorylation docking sites compared to all other S/T phosphorylation sites. At the protein level we found that 14-3-3 docking phosphosites had significantly higher mean disorder and observation frequency compared to all other S/T phosphosites  the docking site phosphorylations, giving a total of 327 phosphorylation sites across 234 proteins , which was shown by Madeira et al. to outperform other tools for 14-3-3 site prediction.58 14-3-3Pred provides 3 different model outputs: an Artificial Neural Net (ANN), a Support Vector Machine (SVM), and a Position-Specific Scoring Matrix (PSSM). These outputs focus mainly on the amino acids within the binding region and their position relative to the pSer/Thr. Using these outputs, 14-3-3Pred defines sequence-matching cutoffs above which the score for a given site is classified as “high”, indicating a possible 14-3-3 docking site. Therefore, in total we used five different inputs for training our model: the ANN, SVM, and PSSM from 14-3-3Pred, a disorder score from IuPred2A, and a PTM score that we derived from PhosphoSitePlus.56 The PTM score reflects the frequency of unbiased LC-MS/MS-based observation for a given PTM. Importantly, because MS-based detection of PTMs is influenced by numerous factors  and some proteins may be heavily phosphorylated while others are not, we generated a score to represent the PTM observation frequency normalized to the total number of phosphorylations on a given protein (equation below), providing a “PTM score’ that allows for fairer comparisons between different proteins.Next we asked whether integrating MS PTM observation frequency and/or the degree of intrinsic disorder around the PTM site with consensus sequence matching could improve predictions of 14-3-3 docking site phosphorylations. We took advantage of current prediction technology for 14-3-3 docking sites by using the web-based application developed by the Barton and MacKintosh groups called 14-3-3Pred , which we call “14-3-3 site-finder”, the first output is “Total Score”, which accounts for all 5 inputs . To visualize the relative contributions of the different inputs, we also generated random forest-modeled output columns for “PTM score and Disorder” and “14-3-3Pred scores” separately. We generated a fourth column, termed “Adapted 14-3-3Pred Score”, by using the specified cutoffs from 14-3-3Pred consensus scores, an average of the ANN, PSSM, and SVM scores, and adapting them into a rank order list for comparison to our other outputs within the tool.We trained our algorithm using a variety of arrangements of the 5 different variables, giving our tool 3 different outputs, all of which form a list of predicted 14-3-3 docking sites from most likely to least likely. Within the app  toward sites that closely match the historical consensus sequence for 14-3-3 docking sites.To identify non-canonical 14-3-3 docking sequences, we looked for proteins within our training data set (known 14-3-3 binding sites) that had the lowest consensus sequence scores, defined as any that fell below the 0.5 cutoff score of 14-3-3Pred. This gave us 68 14-3-3 docking site phosphorylations. Then we removed each of these sites from our training data set, retrained the tool, then allowed the tool to predict de novo the 14-3-3 docking sites on each protein in this set individually. We then set a generous cutoff, asking whether the 14-3-3 docking site was identified within at least the top 15 sites predicted by the tool. Interestingly, we found that for the small minority of proteins that lack a PTM score , they consistently failed to rise above the top 15 cutoff, emphasizing the importance of the PTM score in instances where the 14-3-3 docking site phosphorylation is a poor match to consensus sequence. We then selected the lowest ranked  5 docking sites that also had PTM scores for further analysis. These were estrogen receptor-1 (ESR1) pS294, platelet glycoprotein Ib alpha chain (GP1BA) pS607, phosphatidate phosphatase LPIN1 (LPIN1) pS607, serine/threonine-protein kinase Chk1 (CHEK1) pS296, and Mitogen-activated protein kinase 7 (MAPK7) pS486. All five 14-3-3 docking sites on these proteins were predicted within the top 6 sites on each respective protein by the PTM & disorder score from our tool  as well as a less stringent subset of non-canonical sites the fell below a 0.8 cutoff in consensus score. However, these tools failed to outperform the tool trained with the full set of client proteins in identifying non-canonical sites , perhaps69 In this structure, the phospho-peptide adopts a kinked/u-turn conformation at the +2 proline. We also used a co-crystal structure (PDB: 3MHR) of 14-3-3σ in complex with YAP pS127-peptide (RAHSSPASL)64 in which the phospho-peptide adopts a more extended/unkinked conformation  as shown in 31 including Lys47, Arg56, Arg129, and Tyr130, are indicated in green. Our modeling suggests that these non-canonical sequences may adopt an extended/unkinked conformation within the 14-3-3 binding groove.To examine these non-canonical 14-3-3 docking sites in more detail, we used structural modeling of 14-3-3 in complex with the docking site phosphorylation in question. As a homology model, we used the co-crystal structure (PDB: 1YWT) of 14-3-3σ in complex with a mode 1 phospho-peptide (MARSHSYPAGKK).ormation , serving71 which motivated the development of this tool. One of these 14-3-3 client proteins is the non-receptor tyrosine kinase TNK1, which we ultimately found to interact with 14-3-3 through a phosphorylation at S502 within the unusual docking sequence of ISRpSLES.71 As shown in In our previous work, we identified several 14-3-3 client proteins whose docking site phosphorylations were difficult to identify,72 For most experimentally identified 14-3-3 client proteins, only one docking site phosphorylation is known,49 yet it is likely that phosphorylations at a second site contribute to 14-3-3 binding on those proteins. In searching the list of 14-3-3 client proteins with only one identified docking site phosphorylation, we identified AKAP13 as an interesting candidate for identification of a second docking site phosphorylation. Diviani et al. used Scansite, an older iteration of 14-3-3 docking prediction software, to identify S1565 as required for 14-3-3 binding and found that 14-3-3 binding inhibits AKAP13 Rho-GEF activity.73 However, we found that AKAP13 has long regions of disorder with numerous phosphorylations, many of which are identified more frequently than S1565 in unbiased MS studies  for the PTM and 14-3-3 research communities.A major challenge to PTM research is sifting through the reams of PTM proteomics data for biologically functional protein modifications, which often exist among numerous modifications that have seemingly little to no impact on protein function. In our view, the 14-3-3 interactome has the potential to accelerate this search, guiding us to phosphorylations that control core biological functions. However, in our experience, the most costly and time-consuming step has been identifying 14-3-3 docking site phosphorylations, often among numerous phosphorylation sites with similar sequences on a given protein. Our data suggest that combining the easily extractable protein features of intrinsic disorder and PTM observation frequency accelerates the identification of 14-3-3 docking site phosphorylations. We have now combined these features with consensus sequence prediction from 14-3-3Pred in a web app  or RXF/YXpSXP (mode II). Other groups have discussed in detail how these non-canonical 14-3-3 docking sequences have become relatively common,75 Furthermore, phosphorylations with high MS observation frequency are likely abundant, high stoichiometry phosphorylations, which logically tend to be functionally important.76 Interestingly, our observation that 14-3-3 docking site phosphorylations are among the most observed phosphorylations on a given protein may refelect the role of 14-3-3 in protecting phosphorylations from phosphatase activity, which would naturally increase the MS observation frequency. This function of 14-3-3 has been demonsrated for a handful of client proteins, but is perhaps more common than we appreciated.62 Additionally, other protein structural or sequence features could be harnessed to improve 14-3-3 docking site predictions. For example, the structure of 14-3-3 bound to BRAF, as well as other 14-3-3 client protein co-structures, highlight contact sites that are distant from the 14-3-3 phospho-binding groove.78 However, fully realizing the predictive power of these additional features is limited by our incomplete structural understanding of many 14-3-3 client proteins, further complicated by the fact that many docking sites sit within disordered regions that are difficult to resolve by structural techniques.Our results raise questions about why intrinsic disorder and PTM observation frequency are predictive for 14-3-3 docking site phosphorylations. We noted that for many 14-3-3 client proteins, there are long stretches of disorder on both sides of the phosphorylation. These disordered stretches likely help accommodate phosphorylated proteins into the rigid structure and phospho-binding grooves of 14-3-3, which are ~34 Å apart.81 to therapeutically modulate 14-3-3 interactions.In summary, this study provides a new tool to accelerate the discovery of 14-3-3 docking site phosphorylations, validated by the identification of sites that are poor matches to consensus sequence and the identification of a novel 14-3-3 docking site phosphorylation on AKAP13. Future work will focus on identifying additional features predictive for 14-3-3 binding. Understanding the full scope of what mediates 14-3-3 interactions will provide insight into how the specificity of 14-3-3 binding is controlled, and perhaps even the determinants of 14-3-3 isoform-specific interactions—a long standing question in 14-3-3 research. Ultimately, understanding how 14-3-3 regulates client proteins within its vast and still largely untapped interactome will continue to provide critical insight into fundamental biological processes and expand possibilities for promising new approaches, including molecular glues,Supplementary figure 1Supplementary table/dataset 2Supplementary table/dataset 1


Coronavirus papain-like proteases (PLpros) play a crucial role in virus replication and the evasion of the host immune response. Infectious bronchitis virus (IBV) encodes a proteolytically defective remnant of PL1pro and an active PL2pro. However, the function of PL1pro in IBV remains largely unknown. This study aims to explore the effect of PL1pro on virus replication and underlying mechanisms.The recombinant viruses rIBV-ΔPL1pro and rIBV-ΔPL1pro-N were obtained using reverse genetic techniques through the deletion of the IBV PL1pro domain and the N-terminal conserved sequence of PL1pro (PL1pro-N). We observed significantly lower replication of rIBV-ΔPL1pro and rIBV-ΔPL1pro-N than wild-type IBV. Further investigation revealed that the lack of PL1pro-N in IBV decreased virus resistance to interferon (IFN) while also inducing host immune response by enhancing the production of IFN-β and activating the downstream STAT1 signaling pathway of IFNs. In addition, the overexpression of PL1pro-N significantly suppressed type I IFN response by down-regulating the expressions of genes in the IFN pathway.Our data demonstrated that IBV PL1pro plays a crucial role in IBV replication and the suppression of host innate immune responses, suggesting that IBV PL1pro could serve as a promising molecular target for antiviral therapy.The online version contains supplementary material available at 10.1186/s12917-023-03839-2. Gammacoronavirus. Although it has been recognized since the 1930s, IBV remains a primary cause of disease and economic losses in the poultry industry [Coronaviruses are significant pathogens causing respiratory diseases in humans and many animal species . Avian iindustry . IBV is industry –6.Alphacoronavirus and clade A of Betacoronavirus [Alphacoronavirus transmissible gastroenteritis virus (TGEV) has been determined [Papain-like protease domains in the nsp3 of coronaviruses are essential for releasing nsp1, nsp2, and nsp3 from the N-terminal region of polyproteins. Besides their function in the viral replicase polyproteins processing, PLpros also play a crucial role in antagonizing interferon (IFN) response through their deubiquitinase (DUB) activity –10. Whilonavirus . Only ontermined . The strtermined . TGEV PLtermined . Porcinetermined . Given ttermined , 16.Gammacoronavirus IBV encodes a proteolytically defective remnant of PL1pro and an active PL2pro, currently known as PLpro. The PL1pro in IBV is incomplete due to the absence of the zinc-finger motif and the catalytic triad residues [The residues . Why hasThis study utilized the IBV-Beaudette strain (IBV-P65) to investigate the involvement of PL1pro in IBV replication and the underlying mechanisms. PL1pro of IBV-P65 is located between two conserved functional domains: the Ac domain (Glu-rich acidic domain) and the macrodomain , with a length of 519 nucleotides (3022–3540 nt), which encode 173 amino acids. Sequence alignment of PL1pro from different IBV isolates  showed a high level of conservation in the N-terminal region (PL1pro-N) with 64 amino acid residues and in the C-terminal region (PL1pro-C) comprised of 12 amino acid residues (homology 92%–100%). However, the region between these two has significant variability in the amino acid residues  spanning the entire IBV genome via RT-PCR. These fragments were derived from Vero cells infected with the IBV Beaudette strain (p65) . These fRT-PCR analysis of IBV subgenomic mRNA 2 was conducted to confirm the virus replication. The recovery of rIBV-ΔPL1pro and rIBV-ΔPL1pro-N from the in vitro-synthesized full-length transcripts was confirmed by sequencing the RT-PCR fragments that covered the deleted regions , which inhibit viral replication through various mechanisms . PhosphoThe IFIT (interferon-induced proteins with tetratricopeptide repeats) family are prominent ISGs regulated by either viruses or interferons. IFIT proteins have been identified as potent antiviral proteins . To analTo verify the regulatory effect of IBV PL1pro-N on the type I IFN pathway, we constructed plasmid encoding IBV PL1pro-N and overexpressed IBV PL1pro-N in H1299 cells. Western blot was performed to investigate the expressions of IFN-β, p-STAT1, IFIT1, and IFIT2 in cells. As shown in Fig. The Beaudette strain of IBV, grown in chicken embryos, was adapted to Vero cells through serial passages, and later this Vero cell-adapted IBV Beaudette strain can also infect human cell lines H1299 and Huh . In our E. coli. To ensure the accuracy of the genomic sequence, we perform sequencing on each IBV cDNA plasmid before initiating virus recovery in our reverse genetic system. Another factor that impacts the success of a reverse genetic system is the lower efficiency of electroporation. During the virus recovery process, we observed variability in the electroporation efficiency of Vero cell lineages. Therefore, enhancing electroporation efficiency and increasing the success rate of reverse genetics are essential challenges that need to be addressed in the future. Addressing these challenges will enable a broader utilization of these techniques for studying viral protein function.To our knowledge, this is the first study to elucidate the effect of IBV PL1pro on IBV replication and host innate immune response using a reverse genetic system. The reverse genetic system allows for the production of infectious viruses from cDNAs. This system can be used to adeptly modify viral genomes, facilitating research into the roles of viral proteins in virulence, innate immunity, and both in vitro and in vivo antiviral drug testing , 28. CurCoronaviruses encode either a single or two PLpros, known as PL1pro and PL2pro, which are significant subdomains of nsp3 , 34. AltThe innate immune system, which consists of immune cells, immune tissues, and immune organs, plays a crucial role in the initial detection and restriction of pathogens . HoweverPrevious studies have also shown the resistance of IBV to IFN treatment and further demonstrated that the resistance has been ascribed to the ability of IBV to inhibit the phosphorylation of STAT1 and nuclear translocation of p-STAT1 . Given tIn summary, our study demonstrated the effect of IBV PL1pro on IBV replication and host innate immune response. The results showed that IBV PL1pro and the N-terminal region of PL1pro were involved in virus replication. In the following underlying mechanisms investigation, we found that IBV PL1pro-N was responsible for the observed IFN resistance by IBV. In addition, IBV PL1pro-N regulated IFN production and signaling pathways downstream of IFN Fig. .◦C in 5% CO2. IBV Beaudette strain (GenBank accession number: DQ001339) was preserved in our laboratory . The recombinant viruses rIBV, rIBV-ΔPL1pro, and rIBV-ΔPL1pro-N were constructed in this study by reverse genetics, as further detailed below. Plasmids pKTO-IBV-A, pGEM-IBV-B, pXL-IBV-C, pGEM-IBV-D, pGEM-IBV-E bearing IBV Beaudette fragment A, B, C, D, and E covering the full-length genome and plasmid pKTO-IBV-N containing N gene and 3’-UTR were preserved in our laboratory.H1299 cells were purchased from Procell Life Science&Technology Co.,Ltd (China). Vero cells were provided by American Type Culture Collection (ATCC No.: CCL-81). Both cell lines were maintained in Dulbecco’s Modified Eagle Medium  and supplemented with 10% fetal bovine serum , 100 µg/mL streptomycin and 100 U/mL penicillin  at 37Plasmid encoding IBV PL1pro-N was generated by amplifying cDNA from pKTO-IBV-A using the corresponding primers and cloning it into pcDNA5-FLAG. Plasmid pKTO-IBV-ΔPL1pro and pKTO-IBV-ΔPL1pro-N were generated by deletion of the PL1pro domain and the N-terminal region of PL1pro on pKTO-IBV-A using PCR. The constructed plasmids were confirmed by sequencing the whole fragments. Table H1299 cells were transfected with the overexpression plasmid pcDNA5-FLAG-PL1pro-N using TurboFect Transfection Reagent  following the manufacturer’s instructions when they reached 80% confluence in a 6-well plate. After 24 h of transfection, Western blot was used to detect the overexpression effect.The wild-type recombinant IBV (rIBV) was constructed by reverse genetics, as described previously , 31. Ini5 cells/well one day before infection. Serial dilutions of each viral stock supernatant were prepared and used for inoculation into Vero cells. After incubating for 2 h at 37℃, the inoculum was removed. Cells were subsequently overlaid with 0.4% agar in FBS-free DMEM, incubated at 37℃ for 3–4 days. After incubation, the cells were fixed with 10% formaldehyde for 10 min and stained with a 0.2% crystal violet solution following the removal of agarose. The plaques were counted, size was measured, and virus titers were calculated according to plaque-forming units (PFUs).Vero cells were seeded into 6-well plates at 5.0 × 10Vero and H1299 cells were infected with rIBV, rIBV-ΔPL1pro, and rIBV-ΔPL1pro-N at a MOI of 0.5. The viral stocks were collected at 0, 4, 8, 16, 24, 36 and 48 h.p.i. TCID50 determined the viral titers: the stocks were serially diluted 10-fold with FBS-free DMEM inoculated to 80% confluence of cells in 96 well plates and incubated at 37˚C for 3–4 days. The CPE was observed 3–4 days after infection, and the TCID50 was calculated using Reed and Munch mathematical analysis.H1299 cells were seeded in a 6-well plate and allowed to reach 80% confluence. Before infection, they were pretreated with varying concentrations of recombinant human IFN-  for 2 h. The cells were infected with recombinant viruses rIBV and rIBV-ΔPL1pro-N at a multiplicity of infection (MOI) of 0.5. At 18 h post-infection, virus replication was detected using indirect immunofluorescence assays targeting the IBV N protein.RNAs were extracted from H1299 cells using TRIzol reagent  according to the manufacturer’s instructions, and RNA concentrations were determined using an OD1000 instrument. The isolated RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent kit with gDNA Eraser  following the manufacturer’s instructions; then, cDNA served as a template for RT-PCR and RT-qPCR. The relative RNA levels in each sample were normalized using cellular GAPDH mRNA. The primers for PCR are shown in Table Cell lysis was conducted using RIPA lysis buffer, and cell debris was removed by centrifugation at 12,000 × g for 10 min. The supernatant was mixed with SDS sample loading buffer and subsequently subjected to electrophoresis on 10% or 12% polyacrylamide gels. The separated proteins were transferred onto polyvinylidene fluoride (PVDF) filter membranes . The membranes were blocked with a blocking buffer (5% skimmed milk in TBS with 0.1% Tween 20) at room temperature for 2 h and then incubated with primary antibodies overnight at 4˚C following dilution. After incubation, membranes were washed three times with washing buffer (0.1% Tween in TBS) and incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody at 37℃ for 2 h. Finally, the samples were detected using a chemiluminescence detection kit following the instructions provided by the manufacturer. The antibodies for Western blot were as follows: anti-IBV-N antibody , rabbit anti-IFN-β polyclonal antibody , rabbit anti-STAT1 polyclonal antibody , rabbit anti-IFIT1 polyclonal antibody , rabbit anti-IFIT2 polyclonal antibody , rabbit anti-phospho-STAT1-Y701 antibody , HRP-conjugated goat anti-rabbit IgG .H1299 cells were cultured in a 24-well plate until reaching 80% confluence and then infected with rIBV and rIBV-ΔPL1pro-N at an MOI of 0.5. Cell culture was discarded at 24 h post-infection. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 minutes, followed by permeabilization with 0.5% Triton-X100 for 15 minutes at room temperature after being washed three times with PBS. The plate was blocked using 5% FBS at room temperature for 1 h, then incubated with anti-IBV-N antibody (1:200) and rabbit anti-phospho-STAT1-Y701 antibody (1:100) at 4 ºC overnight. After washing, the tissue samples were incubated with FITC-conjugated goat anti-rabbit IgG  at room temperature for 1 hour. Nuclei were stained with 4’, 6-diamino-2-phenylindole (DAPI)  for 15 min. Finally, the images were captured using a fluorescence microscope .Statistical analysis was conducted using SPSS 23.0, while graphs were created using GraphPad Prism 8. The intensity of protein bands was quantified using ImageJ software. The data were represented as mean ± standard deviation (SD) of three independent experiments. The significance between groups was determined using the student’s t-test. P values < 0.05 were considered statistically significant.Below is the link to the electronic supplementary material.Supplementary Material 1


Overdoses are increasing in the province of Ontario, Canada, where northern communities such as Sudbury have witnessed disproportionately elevated rates, with opioid-related deaths double that of the provincial average. To address this issue, governments have implemented supervised consumption services (SCS) where people who use drugs (PWUD) can use their pre-obtained substances onsite under trained supervision. In September 2022, the city of Sudbury opened its first SCS, ‘The Spot’, but the site’s sustainability is contingent on demonstrating benefit to PWUD and the neighboring community. We undertook a qualitative study exploring experiences among clients who used the consumption service inside The Spot. In December 2022, clients of The Spot were invited to participate in a brief survey which collected socio-demographic information and substance use profiles, followed by an in-person semi-structured qualitative interview. Participant survey and interview data were combined with administrative site utilization data provided by site staff of all clients who accessed the consumption service from September 2022 to August 2023 to examine overall service utilization and uptake. Qualitative data were analyzed using iterative thematic analysis techniques, and results were informed by common responses to research questions. The responses were narratively presented. Administrative site utilization data highlighted a relatively stable increase in uptake and utilization of the site since its inception. A total of 20 clients participated in the survey and semi-structured interviews. Participants described the importance of the site in preventing and responding to overdoses, providing a safe and comfortable environment to consume their drugs, and decreasing public drug use, which they suggested may potentially reduce stigmatization in the community. However, clients also suggested challenges, including issues regarding site operational policies that hindered consumption room utilization. Service suggestions made by clients to improve site utilization include the addition of inhalation services, relocating the site to a location in downtown Sudbury where PWUD commonly congregate, and extending operational hours. Positive impacts and recommendations can be drawn on and considered by other northern or rural communities interested in implementing similar harm reduction services. Substance use and related harms have sharply increased across Canada over the past decade, and especially following the onset of the COVID-19 pandemic , 3. DataTo address this ongoing crisis, governments across Canada and within Ontario have implemented several harm reduction measures such as supervised consumption services (SCS), with thirty-eight sites operational in Canada as of 2023, twenty-six of which are located in Ontario, and two of which are located in Northern Ontario  . SCS areAlthough the literature on the benefits of SCS is well established and demonstrates many benefits for PWUD and their communities, these services are less available and accessible in northern and rural jurisdictions in Ontario despite the disproportionately elevated overdose rates in many of these communities , 4. HistThe city of Sudbury, Ontario, is one such example of a community that has faced many challenges in their efforts to apply for a permanent SCS, underscoring the difficulties some Northern Ontario communities have endured in implementing a SCS. To meet application requirements and corresponding permanent site approval, the CDS undertook a needs assessment between 2019–2020 which consisted of a survey with 190 people who inject drugs (PWID), an online survey with 2,251 community members, focus groups with 52 community partners and stakeholders, and secondary data analysis . The assFollowing the needs assessment and the subsequent submission of a SCS application, in May 2022, Sudbury was granted a time-limited federal exemption from Health Canada to operate the community’s first federally sanctioned SCS. The temporary site, also known as ‘The Spot,’ opened on September 28, 2022, and provides an array of services such as distribution of harm reduction supplies, consumption services, and referrals to other social and health services. The site is run by the Réseau ACCESS Network, a non-profit, community organization committed to holistic and comprehensive approaches in promoting wellness, harm and risk reduction, and education, which operates in a different location than The Spot . AlthougGiven the novelty of the service in the City of Sudbury after longstanding harms associated with drug use, and lack of certainty regarding the sustainability of the site, it is important to explore how the site is being experienced by PWUD within the community, including perceived benefits, challenges, and impacts. Gaining an understanding of such experiences and perceptions will be important to understand the site’s utility and ability to meet community needs. As such, we conducted a study to qualitatively examine client experiences of using the consumption service at The Spot through in-depth semi-structured interviews. This data were complemented by administrative site utilization data provided by The Spot staff which included overall uptake of the consumption service based on total number of visits and consumptions, as well self-reported substances consumed within the consumption room by clients. The results of the study can be used to demonstrate the benefits and challenges of the site as well as to inform decision-making practices regarding the sites’ sustainability and impact, including evidence of the community’s need for site permanency.The study involved a brief in-person socio-demographic survey followed by a qualitative semi-structured one-on-one interview. The survey collected basic socio-demographics including the age, gender, ethnicity, living situation, and substance use profiles of each participant. The qualitative semi-structured interview guide was developed in collaboration with the primary research team and peer advisors. The interview questions explored participants’ initial perceptions of the site, including any benefits, challenges, and barriers to accessing the site, as well as any impact it has had on, or will potentially have on, drug use patterns and risks of associated harms, and whether, or to what extent, the site is meeting the needs of PWUD. In addition, aggregate site utilization data between September 28, 2022 and August 31, 2023 were provided by the site staff which included monthly totals of the number of consumptions, visits, new clients, as well as the number and types of drugs consumed by clients who accessed the consumption service to contextualize overall consumption room utilization.Clients were eligible if they were: aged 18 years or older, currently living in Sudbury, currently using illicit substances, fluent in English, and had used the consumption service at The Spot at least once. Clients who attended the site only for harm reduction supplies or did not speak English were excluded from the study.A sign-up sheet that included three days to participate in a scheduled interview (with 60-minute time slots) was circulated by staff members two weeks in advance of data collection. Staff assisted with recruitment by posting recruitment flyers at consumption booths and spreading the word to clients via word of mouth. Interested clients signed up at a specified time slot. In addition, the research team allocated two additional drop-in days where consumption room clients could participate on a first come first served basis as they accessed the consumption room, with no prior appointment needed.The study was conducted between December 1 and 5, 2022 in a private room at The Spot, which allowed two trained members of the research team (FA and CR) to conduct the interviews. The research team collected written informed consent from participants prior to commencing the study. Clients were asked to participate in a survey lasting about five minutes whereby demographic characteristics and substance use profiles were captured via an online tool (Research Electronic Database Capture [REDCap]). A 30-minute, audio recorded, one-on-one qualitative semi-structured interview was subsequently conducted, which asked clients about their experiences and perceptions of the site. Study participants were assigned a unique code to maintain their confidentiality. To compensate clients for their time and participation, $30 cash honoraria was provided upon completion of the study. The study was approved by the Centre for Addiction and Mental Health (CAMH) Research Ethics Board (#087/2022). From September 2022 to August 31, 2023, aggregate site utilization data were recorded monthly on Excel documents by staff at the site and shared with the research team via a Microsoft word document that provided monthly breakdowns of the total number of consumptions, visits, new clients, as well as the number and types of drugs consumed by clients who accessed the consumption service. Number of overdoses that were reversed at the site were also included in the data . The site utilization data were collected by staff and clients via an intake form which was completed with every client prior to entering the consumption room (regardless of whether it was their first visit or not). Data on the type of drug clients intend on consuming in the room, based on what they believe they have purchased  was also collected.All quantitative survey data  were exported into Excel from REDCap. For both the quantitative survey data and the aggregate monthly site utilization data provided by staff, basic descriptive statistics were applied.All qualitative data audio files were uploaded onto a secure network, and later transcribed verbatim via a third-party company, with all identifying information removed. The transcripts were reviewed for accuracy, and imported into qualitative software . An initial codebook of themes was prepared based on the interview guide, research questions, and preliminary analyses . The transcripts were reviewed by two members of the research team (FA and CR) who identified and coded common themes. The codebook was subject to further development and revision based on ongoing analyses and discussion of emergent themes among members of the research team. All qualitative data were analyzed using iterative descriptive thematic analysis techniques, informed by Braun & Clarke’s (2006) six phases of thematic analyses . Themes The first section of the results reports overall usage of the consumption room, based on administrative aggregate site utilization data retrieved from The Spot. This was used to identify overall uptake of the consumption room in addition to client substance use profiles. The second section of the results highlights participant data from the survey, documenting their socio-demographic characteristics and substance use profiles. This is followed by the qualitative findings.Between September 28, 2022, and August 31, 2023, a total of 470 unique clients accessed The Spot, representing 1,181 total visits, and 1,605 total consumptions see . All sitOf the drugs reported, fentanyl was the most commonly self-reported injected drug with 1,005 consumptions, followed by speedballs  (n = 286 consumptions), and methamphetamine (n = 111 consumptions). The Spot has reversed a total of 20 overdose events, nine of which required naloxone, the remaining seven required oxygen only as the intervention. No overdose required EMS.Table 1). The average age of participants was 37, with the majority aged 31–50 . Nearly three-fourths identified as men , and half  identified as Indigenous. Almost half  reported experiencing homelessness.A total of 20 clients participated in the study , followed by crack cocaine , and methamphetamine . In terms of routes of consumption, the most common route of illicit opioid consumption was injection  followed by inhalation . Of the 17 participants who used crack cocaine, the most common route of consumption was inhalation  followed by injection . Of the 15 people who used methamphetamine, injection was the most common route of consumption , followed by inhalation . See The findings from the study are presented narratively below and organized into the following thematic categories: 1) Perceived impacts on substance use, including perceptions of overdose risk and frequency of substance use; 2) Perceived impacts on stigma, including community-level stigma, and 3) Perceived operational impacts, including existing policies , location, and hours of operation. The themes are illustrated with select quotes from participants, where appropriate.Perceptions of overdose risk. The most common benefit of the site identified by participants related to increased feelings of safety when using the site to inject their substances. All participants stated that they felt safe using their substances in the consumption room where their use and any potential adverse effects were monitored by trained staff in proximity. This was particularly relevant for participants who expressed a fear of overdosing  and suggested they felt at ease knowing that if they were to overdose, there would be trained staff present to intervene. For instance, when asked why they decided to come to The Spot, one participant described:“For safety. Because I would just prefer to be somewhere where I’m safe and know If I do overdose, there’s someone there to help me… I’m here for that safety reason like I said, I don’t want to die. Bottom line.”I almost died a couple times, and my friends I was with at the time just left me there, you know what I mean? They won’t let me die in here.”Some participants specifically described the importance of having trained staff available on site when using their drugs as a means to prevent and respond to overdoses, as they indicated they could not trust their friends or peers to intervene in the event of an overdose. Some participants provided anecdotes of situations they had been in where the people they were using with had left them during an overdose: “Another participant reiterated this sentiment, and suggested justifications for why people typically leave the scene when someone overdoses, including a fear of criminalization or because they did not want to ruin their own high, which would not happen at the site:“A lot less people aren’t going to lose their life because of [The Spot]. It makes me sick because I’ve been around so many people that have overdosed and everyone just leaves because they’re scared to get involved with the police and say “oh it’s a buzz kill.”“Just to be in a safe environment, and if I’m using by myself and I come here at least I don’t have to worry about overdosing.”Participants who disclosed that they typically used alone  also reiterated the importance of being able to use under the watch of staff as an important overdose mitigation and lifesaving strategy: Frequency of substance use. Although the site had only been open a short while, participants discussed the positive impacts the site could potentially have on the frequency of their substance use long-term. Half  of participants suggested that if they limited their drug use to only injecting when they are at The Spot, it could help them reduce their use:“It’d kind of help me slow down. Like if I chose to just do it here…[I would use less]… and if I wanted to just quit, that way I won’t go anywhere else like on the street or you know, like my place of residence by myself, you know? [I would use the site to slow down and eventually quit].”Participants speculated that the main reasons they might limit their use to only when they were able to access the site was due to increased feelings of safety when consuming there:“I will not inject anywhere else other than here. Not at a friend’s house. Not even with another person if they’re with me. I just won’t. I don’t feel comfortable…there’s many times when I won’t inject because they’re closed.”However, while some participants suggested The Spot could potentially support reductions in drug use, two participants indicated the opposite, and described the possibility for their use to increase due to the same feelings of safety when consuming at the site, and the assurance that they would be attended to in the event of an overdose.The presence of stigma against drug use and PWUD within the Sudbury community was commonly discussed by participants. Participants suggested that the longstanding visibility of public consumption and subsequent paraphernalia littering had prompted negative community attitudes and perceptions towards PWUD and had reinforced stigma against them. These attitudes were heightened due to the implementation of The Spot, where some participants  discussed how the site was not supported by many Sudbury residents and business owners—many of whom felt as though the site enabled drug use:“There’s a stigma attached to this place for sure. I personally don’t care. Well I can’t say that. I do care what people think. I think no matter where this building is there’s always gonna be negative people. Saying you’re enabling them. But it’s an epidemic, people are dying, they need places like this to keep people alive… I’ve had three people die over the weekend of overdoses, because they were using outside by themselves.”One of the main issues related to community-level stigma as described by participants was the increased visibility of public drug use. Participants discussed how public drug use had become entrenched and normalized, particularly within the downtown Sudbury area, and participants disclosed that they were frustrated and sad about this reality:“Five years ago you could walk around, there wasn’t needles everywhere. Now, it’s like every corner you go by there’s a needle, an uncapped needle on top of that. Even parks where kids play, I found a few of them sitting there. I’ll grab them and pick them up and put em in my pocket and go discard them because it’s not fair for a kid. Like what if they take off their shoes and run in the sand and get pricked by a needle and what, they get sick, they get AIDS, something like that? That’s not cool.”However, participants speculated that the presence of the site could potentially work to mitigate the visibility of public consumption, which could reduce community-level stigma towards them. Participants suggested that if the site worked to encourage people to only use there, where they can safely discard their used equipment and use away from the public’s gaze, that this could potentially alleviate community concerns:“It’s gotten to be too much of nothing for drug users to be walking around and probably nodding off and there’s a good chance they could be dead and [pedestrians] just walk right by. If they’re [at The Spot] it’s good for a lot of people, the people who don’t die, the people who don’t have to see it, kids.”Some participants elaborated on the site’s potential to not only reduce public consumption, but to also increase community safety via less littered paraphernalia and discarded needles. Participants stressed the potential for the site to be able to reduce the number of discarded needles, and that this could be a major benefit to the community:"What would they [Sudbury community] rather have? More people laying on the sidewalk or more needles downtown? Anyone who doesn’t want this [The Spot] here doesn’t get the benefits of it. You get more people sleeping in daylight, in front of business, shop owners having to clean needles out of their front every day. Even the closed-minded people have to get that."However, just over a third of participants  suggested that community-level stigma was so engrained within the community that the site would likely not make a difference:“[The stigma] has gotten so bad in this city. The people who don’t use are just like, oh, another one. You can learn ten thousand great things about me, but the second you learn I’m a junkie, that’s it. I don’t think we’ll ever get rid of that. There’s always gonna be a stigma attached to someone.”Peer harm reduction staff involvement. Among the positive attributes of the site that were discussed by participants, the presence of peer harm reduction workers with lived and living experience with drug use was noted as an invaluable asset. Participants emphasized that the presence of these workers provided them with comfort and suggested that this contributed to fostering a non-stigmatizing and non-judgmental environment. Knowing that staff had extensive knowledge about drugs made PWUD feel more comfortable as they could relate to them:“Well, the one guy, he’s been where I am, so he knows. I don’t know exactly where he’s been, but I know [he has lived experience]. Like hands-on. He’s given me advice on stuff. Changed my life talking to him and stuff.”For some participants, the presence of peer harm reduction workers was considered inspirational, as they saw themselves reflected in the workers and were motivated by the idea that people in their shoes could possibly stabilize and manage their drug use and attain employment in a field where they are knowledgeable and passionate:“It’s nice to see some of the local people here. They’ve got drugs. They’re working. They’re off the street. They look clean and fresh and they’re not using on site, I can tell. They’re holding it down. Kudos to them. To get up and go to work and hold it down all day.”Lack of inhalation services. A commonly discussed issue with the site was that inhalation services where clients could smoke their drugs were not offered or available and inhalation on site was not permitted. Most participants  both injected and smoked their substances, some of whom identified a preference for inhalation . As such, the majority of participants  expressed the importance of inhalation services and suggested that the lack of inhalation services was a potential deterrent to accessing the site. Participants further noted confusion regarding the ability to inject but not smoke their substances, and given the dangers of inhaling substances, did not understand the rationale behind this policy:“If you’re allowed to shoot up here, you should be allowed to smoke up. I understand the cigarette part, but having a separate room, well ventilated and let the people smoke their fentanyl. You can come up here and shoot crystal meth but you can’t smoke it? Same with the down? You can’t smoke the down but you can shoot it? You should allow it because you can just die just as easy and no one’s there to help you. I wonder why they don’t allow it. That’s a bullshit policy.”Participants further expressed that because the site does not offer inhalation services, The Spot is therefore not able to support a large percentage of the population of PWUD considering many inhale their substances. Furthermore, some participants described how the risks associated with inhalation are just as high as with injection, and suggested that the site therefore does not offer harm reduction and safety for the entire PWUD community:“I’d say half or more of the people smoke [their drugs]. So, you have to take that into consideration too, and how many people have died from just smoking?”“I smoke a bit as a risk mitigation strategy, and I feel it’s safer than injecting. It takes a while to feel the effects of it.”Despite some participants indicating the risks associated with inhalation, many believed that smoking their substances was a safer route of administration compared to injecting. For instance, some participants  who typically consumed their drugs via both injection and inhalation mentioned that they would first smoke their substance before injecting as a harm reduction measure to test the toxicity or to see how strong it was. These participants believed that once they tested their substances via inhalation, they would be able to better determine whether it would be safe to inject: Participants further elaborated that if the site offered a space to inhale, they would use the site more often, including to practice what they considered harm reduction through testing their substances via inhalation prior to injecting:“There’s also a far lower chance of overdosing from smoking, but there still is. A lot of people think you can’t overdose if you smoke it…I think a lot more people would use here [if the site offered inhalation services] because a lot of people do both at the same time…Like I said, I smoke my down before I shoot it, just in case it’s too strong, right? [I would come here to do that test]. With fentanyl, I have watched people with a huge tolerance who do this shit every day, do one puff, not even exhale yet, and they’re dropped. One puff. Now that fentanyl is a big thing like it is, I definitely think [inhalation services] would be a good thing.”“I’d be here every day if they offered inhalation services.”Overall, participants emphasized the importance of having inhalation services and suggested that there would be an increase in site uptake if the site offered such a service: Rules regarding splitting and sharing. Another unfavorable policy was the inability to ‘split’ or ‘share’ drugs with others while in the consumption room. Some participants described that they typically split or shared their drugs with other people for various reasons, including for safety when they do not want to use alone, or when pooling their money together to purchase a larger quantity of drugs than just one could afford, with the idea that they would consume the drugs together. Thus, participants suggested that because the site did not allow this practice, it was not reflective of their use patterns, and was a deterrent to using the consumption room. For example, one participant explained the current policy is not commensurate with how PWUD prefer to use their drugs:“Because you’d maybe want two people to go in, like a couple. I don’t know how that works…for sharing sometimes, because that’s what happens on the street…Yeah, not like anything dirty or anything. But yeah. That would be a thing.”Location. Another commonly reported issue with the site was the location. The Spot is physically located approximately 5 minutes’ drive or a 15-20-minute walk from Sudbury’s downtown core. Most participants  noted that the distance to travel to the site acted as a major barrier to accessing the site. Participants often referred to the site as “out of the way”, and many articulated that the site’s location was situated too far from where they purchased their drugs, which deterred them from utilizing the site. Participants emphasized that the site should be relocated to downtown Sudbury, which would encourage access and uptake of the site:“Preferably I’d like the location to be downtown because that’s where everyone hangs out and that’s where the drugs are. It doesn’t make sense to buy drugs down there and walk all the way up here when you can just shoot it up down there. A lot of people say the same thing.”Participants reiterated they typically need or want to consume their drugs as soon as they get them and are reluctant to travel or walk a long distance to the site. This was particularly relevant for participants who shared experiences of going through withdrawal or feeling ‘dope sick’, and described how during these moments, there is usually an urgency and need to consume their drugs, and that their focus is on not feeling sick as opposed to figuring out where and how they can use more safely. In these instances, many PWUD prioritized relieving their withdrawal symptoms and ensuring they do not feel sick, instead of travelling to the site.:“I guarantee you guys don’t get people here because of the location. I know downtown don’t want it down there and I understand that. But it’s gotta be down there I think. A lot more people would be using this facility if it were closer to the downtown core. Because what do you do? You wake up in the morning and want to do your fix, you’re gonna walk a half a mile first? No…Because it’s the first thing you wanna do when you wake up. You roll over, sometimes sick and the first thing you want to do is get un-sick. If I can walk three minutes compared to 20 minutes, I would be here.”However, nearly a third of participants  noted that they did not mind the location as they either lived nearby, or they preferred that it was outside of the ‘drug scene’, where they were less likely to be recognized or further stigmatized for using the site. For instance, one participant described the importance of the discreetness of the site, and how it made them feel more comfortable and willing to visit it:“I work part-time and if they knew I was using I might not have that job. My buddy here isn’t a user. He knows I do, but I try to hide it from everyone, I really do. I’m not afraid of people seeing me here because it is farther away, right? So the location is good in a way, I never thought of it that way.”“It’s open and there’s no sneaking around. For people who are trying to hide their addiction they’re not hiding jack shit around here.”Similarly, participants discussed potential impacts of the physical infrastructure of the building, particularly in regard to it being physically located in a trailer in an open field. For instance, two participants raised concerns about the lack of privacy of the location, as there were no surrounding buildings or greenery around the trailer. They expressed that this exposure made them feel paranoid or uncomfortable leaving the site, as there was potential to be visible to the public as they could easily be spotted. This raised issues related to the lack of privacy of the location, as described by one participant: “I think most people are paranoid so it’s better that [The Spot]is open. So they can see everything, and no one is hiding.”However, a few participants  described how they appreciated that The Spot was physically located on an open field, as they would have an easier time monitoring their surroundings: Hours of operation. Another concern that participants commonly discussed related to the limited hours of operation. Currently, the site is only open from 10:00am until 4:00pm, seven days a week. All participants suggested that the hours were not long enough and did not reflect the times they typically consumed their drugs. Participants indicated that the site should be open for a longer duration of time during the day to ensure it met their diverse needs:“And the hours, like I said, most people don’t wake up at 10am. They’re usually up at like 7am. I think they should be open earlier and later. What about the nightlife? People go out and start drinking and the next thing you know they wanna do a fix. I know everything cost money too, like the workers. I get that. But if it’s gonna be done, it’s gotta be done right. That’s my opinion.”“Longer hours. I don’t know if it would be possible to have it open 24 hours, you know?”Over a third  of participants suggested it would be ideal for the site to remain open 24 hours a day to accommodate PWUD. For instance, when asked if there was anything that could be improved about the site, one participant stated: “Yeah, well people start withdrawing first thing in the morning, super early. Most people want their shit by 8 o’clock. Just follow the dealers and the street girls.”Participants also specifically discussed the importance of having the site open earlier than 10:00am for a variety of reasons. For instance, another participant reiterated how PWUD typically wake up in withdrawal or feeling ‘dope sick’, and the first thing they need to do is consume their drugs in order to feel better: Other justifications for suggesting the site should open earlier included capitalizing on when people attended the methadone clinic to get their medications, and how many city shelters forced individuals to vacate early in the morning. As such, participants described the importance of having a place to consume their drugs that was accessible at that time:“Well, I know a lot of people, if they’re homeless, they get kicked out. Like that one [shelter], you’re out at 7:00am. And a lot of methadone clinics, mine’s open at 7:30 and the other is open at 9:00. So that’s two hours [before the site opens]. And they need it every day. That’s why there’s dealers down there at that time. Just waiting.”This qualitative study examined client experiences and perceptions regarding Sudbury’s first SCS. Data collected suggest that the site presents considerable benefits to PWUD in Sudbury. Participants discussed the importance of the site’s ability to potentially reduce public use, overall frequency of drug use, and stigma, as well as increase feelings of safety regarding drug use, particularly as it relates to overdose risk. Participants also provided thoughtful suggestions to improve the service, such as the site’s location, hours, and policies related to the lack of inhalation services and inability to split or share drugs.The Spot utilization data further demonstrate a need for the sustainability of the site as there has been a relatively stable increase in uptake and utilization of the site since its inception. The number of consumptions and visits steadily increased month over month until around April, reaching a peak of 202 and 128 respectively. However, the site thereafter experienced a temporary drop in both visits and consumptions, with 118 consumptions and 79 visits in May and 114 consumptions and 77 visits in June. This was followed by a rapid increase in July and August 2023, to numbers surpassing those recorded in the initial months. The number of consumptions and visits in July of 2023 was 224 and 153 respectively and there was 241 consumptions and 201 visits in August of 2023. Research suggests that there may be seasonal impacts on substance use as drug initiation, overdose emergency room visits,police calls for overdoses, and overdoses themselves usually tend to spike in the summer months , 17. In In addition to the site’s ability to reduce overdose-related harms during the ongoing overdose crisis, participants discussed the value of The Spot in its ability to potentially address issues related to public consumption and related community-level stigma. Although these sentiments were largely speculative in nature and any impact will likely be contingent on continued sustainability and uptake of the site, available evidence suggests that SCS can, in fact, reduce public stigma toward PWUD through the reduction of crime rates, drug use, and prevalence of discarded needles in public spaces , 8, 18. Despite these potential benefits, there were a number of suggestions for how the service could be improved. Participants described issues related to the site’s location and operational hours, and suggested that due to its current location, the site is not reaching its full potential. Evidence suggests that travel acts as a significant barrier for PWUD in accessing health and social services , 24, andAnother major drawback noted by participants was the lack of inhalation services within the site. Participants identified that in addition to injecting their substances, they would also inhale, some of whom noted doing so as a harm reduction measure. Many participants within our study expressed perceptions that smoking is safer than injection, with a few reporting that they would engage in smoking their drugs first to test them prior to injecting. Routes of consumption have been associated with distinct risks . For insThe issues discussed among participants indicate the need for The Spot to be reflective of PWUD diverse drug use patterns, needs, and lifestyles, including recognizing the increasingly common route of administration via inhalation , 39. It Due to the small window of time  that the site was open prior to our study, it is likely that the utilization data is an underrepresentation of The Spots’ potential impact. Given this short timeline and the nature of qualitative studies, some responses were speculative and varied depending on whether the participant was discussing their experiences thus far or were sharing their thoughts on the potential impacts the site may have on them and their community in the future. Responses to our questions were also subject to a number of biases inherent in self-report and qualitative data including self-selection, recall and response biases, social desirability bias, etc. Furthermore, our sample is small and experiences  cannot be generalized outside of the specific participants and setting.This initial qualitative exploration of client experiences with Sudbury’s first SCS suggests that the site offers many benefits to PWUD within the community and can reduce fatal overdoses, underscoring its utility as a key harm reduction service that can potentially reduce community-level stigmatization. However, participants described issues regarding site operational policies that currently hinder site uptake and may not achieve the desired impact of the program. Suggestions to improve the site included the addition of inhalation services, relocating it to downtown Sudbury where PWUD congregate, and extending the operational hours. The results of this early study demonstrate the value and need for site permanency.