Chlamydiales species are obligate intracellular bacteria and important human pathogens that have a minimal division machinery lacking the proteins that are essential for bacterial division in other species, such as FtsZ. Chlamydial division requires synthesis of peptidoglycan, which forms a ring at the division septum and is rapidly turned over. However, little is known of peptidoglycan degradation, because many peptidoglycan-degrading enzymes are not encoded by chlamydial genomes. Here we show that an homologue of SpoIID, a peptidoglycan-degrading enzyme involved in sporulation of bacteria such as Bacillus subtilis, is expressed in Chlamydiales, localizes at the division septum, and degrades peptidoglycan in vitro, indicating that SpoIID is not only involved in sporulation but also likely implicated in division of some bacteria. Chlamydiales species are obligate intracellular bacteria lacking a classical peptidoglycan sacculus but relying on peptidoglycan synthesis for cytokinesis. While septal peptidoglycan biosynthesis seems to be regulated by MreB actin and its membrane anchor RodZ rather than FtsZ tubulin in Chlamydiales, the mechanism of peptidoglycan remodeling is poorly understood. An amidase conserved in Chlamydiales is able to cleave peptide stems in peptidoglycan, but it is not clear how peptidoglycan glycan strands are cleaved since no classical lytic transglycosylase is encoded in chlamydial genomes. However, a protein containing a SpoIID domain, known to possess transglycosylase activity in Bacillus subtilis, is conserved in Chlamydiales. We show here that the SpoIID homologue of the Chlamydia-related pathogen Waddlia chondrophila is a septal peptidoglycan-binding protein. Moreover, we demonstrate that SpoIID acts as a lytic transglycosylase on peptidoglycan and as a muramidase on denuded glycan strands in vitro. As SpoIID-like proteins are widespread in nonsporulating bacteria, SpoIID might commonly be a septal peptidoglycan remodeling protein in bacteria, including obligate intracellular pathogens, and thus might represent a promising drug target. N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide units that are cross-linked by peptide bridges. The composition of the peptide bridges can differ among different species but typically contains both d- and l-amino acids. PG synthesis begins in the cytoplasm and at the cytoplasmic membrane to form lipid II. Once lipid II is flipped to the extracytosolic space, the penicillin-binding proteins (PBPs) and/or the proteins of the shape, elongation, division and sporulation (SEDS) family execute the transglycosylation and transpeptidation reactions to generate glycan strands and peptide cross-links, respectively , subsequently directing the invagination of the envelope . PG is aectively –4. PBP a species .Chlamydiales are Gram-negative (diderm) obligate intracellular bacteria, including many important human pathogens such as Chlamydia trachomatis and Chlamydia pneumoniae, which are classified in the Chlamydiaceae family. All members of the Chlamydiales order that are not part of the Chlamydiaceae family are commonly referred to as Chlamydia-related bacteria. They have been much less extensively studied but include putative emergent pathogens such as Waddlia chondrophila  is able to bind PG. SpoIIDWch is septally localized and was shown to be capable of being immunoprecipitated together with RodZ. Furthermore, chlamydial SpoIID, similarly to its B. subtilis homologue, binds PG in vitro and digests denuded glycan chains . However, in contrast to B. subtilis SpoIID (SpoIIDBsu), SpoIIDWch seems to act as a lytic transglycosylase on Gram-negative PG in vitro and as a muramidase on denuded glycan chains, indicating that it might possess dual activities, depending on its substrate. Note that proteins containing a SpoIID domain are present in a large variety of both sporulating bacteria and nonsporulating bacteria, indicating that this domain may fulfill an ancestral role in PG remodeling and cell division, independently of sporulation and before branching of cyanobacteria, firmicutes, and proteobacteria, as already suggested previously by Morlot et al.  of W. chondrophila-infected Vero cells. Sufficient amounts of PG could be obtained and analyzed by the use of advanced and highly sensitive UPLC-QTOF-MS (UPLC-quadrupole time of flight-MS) (W. chondrophila PG peaks were determined using their MS and tandem MS (MS/MS) fragmentation patterns  and in C. trachomatis PG  . The che-Ala M4; , and pea-Ala M4; . All sev-Ala M4;  correspomatis PG . However10.1128/mBio.01128-19.2FIG S1Planctomycetes, Verrucomicrobia, and Chlamydiae) superphylum. Alignments of the orthologs of SpoIID present in the PVC superphylum were performed as described in B. subtilis used in this study are highlighted in red. Download FIG S1, PDF file, 0.2 MB.(A) Structures of the muropeptides listed in Copyright © 2019 Jacquier et al.2019Jacquier et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01128-19.1TEXT S1Text S1, DOCX file, 0.03 MB.Extensive description of the materials and methods used in this article, including strains and growth conditions, quantitative PCR, protein and peptidoglycan isolations, production of antibodies, and bioinformatics analyses. Download Copyright © 2019 Jacquier et al.2019Jacquier et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the W. chondrophila that bind to PG  as a potential candidate interacting with both RodZ and PG.Proteins binding to chlamydial PG might have an important role in the PG biosynthesis/degradation cycle. We recently used mass spectrometry to identify the proteins of nd to PG . We alsodivision . We thusdivision , we perf10.1128/mBio.01128-19.9TABLE S1Table S1, XLSX file, 0.01 MB.Peptides detected by mass spectrometry after coimmunoprecipitation using an anti-RodZ antibody. Download Copyright © 2019 Jacquier et al.2019Jacquier et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the wcw_0967 gene is conserved among Chlamydiales, encoding a protein with an N-terminal signal sequence that might be cleaved (Bacillus subtilis. We thus renamed Wcw_0967 “SpoIIDWch.” SpoIID of B. subtilis (SpoIIDBsu) was shown to bind PG and drive membrane movement during B. subtilis sporulation, simultaneously degrading PG via its lytic transglycosylase activity  at as early a time as 4 h postinfection (p.i.), with a peak 8 h p.i. . SpoIIDWch could be detected in RBs at both 24 and 48 h p.i.  Quantification of 16S RNA and WchspoIID mRNA by qRT-PCR. Results were normalized to the number of bacteria using the qPCR data from panel A. (D) Western blotting using anti-SpoIIDWch on samples harvested at the indicated time points. Equivalent volumes of sample were loaded on all lines. (E and F) PG sacculi of E. coli or V. cholerae were incubated with SpoIIDWch or not incubated with SpoIIDWch (control) overnight at 37°C. The resulting soluble moieties were analyzed by UPLC-QTOF. The identity of the peaks was determined by MS/MS. M4N, anhydro glycan with 4 peptides; ?, uncharacterized PG moiety. (G and H) MS/MS spectra of selected peaks from the data shown in FIG S4, PDF file, 0.1 MB.(A) Detection of 16S DNA at the indicated time points in Vero cells infected with Copyright © 2019 Jacquier et al.2019Jacquier et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the W. chondrophila was investigated using higher-resolution fluorescence microscopy. We observed partial septal localization of SpoIIDWch in dividing RBs at 24 h p.i.  and penicillin-treated (B) cells described in the Wch (green), and anti-W. chondrophila polyclonal antibody staining of the outer membrane (red), and DAPI staining (blue) were performed by the use of ImageJ. Download FIG S2, PDF file, 0.1 MB.Quantification of the localization of SpoIIDCopyright © 2019 Jacquier et al.2019Jacquier et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01128-19.4FIG S3Wch in cells treated with phosphomycin as described for Wch (green), anti-W. chondrophila polyclonal antibody staining the outer membrane (red), and DAPI staining (blue) were performed by the use of ImageJ. Download FIG S3, PDF file, 0.1 MB.Quantification of the localization of SpoIIDCopyright © 2019 Jacquier et al.2019Jacquier et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B. subtilis homologue of SpoIID was shown to bind to PG and to digest naked glycan strands of PG   . IncubatpoIIDBsu   along with other anhydro-muropeptides  2; Bsu, which did not cause accumulation of nonanhydro moieties   and H. Swas used . Finallymoieties . Taken tBsu (Wch. We selected conserved glutamic acid and/or aspartic acid residues (since the two have similar properties) in SpoIID of W. chondrophila, C. trachomatis, and C. pneumoniae  was shown to be essential for the activity of SpoIIDBsu . We wond version  to C. We version . Interesles spp. .10.1128/mBio.01128-19.6FIG S5E. coli expressing the indicated point mutants or the wild-type version of SpoIIDWch was lysed, and the lysate was centrifuged at 20,000 × g for 10 min. Equivalent amounts of pellet and supernatant were separated by SDS-PAGE and subjected to Coomassie staining. (C) PG of E. coli was incubated with the indicated purified recombinant proteins for 30 min on ice. The suspension was then centrifuged at 20,000 × g for 30 min and washed once. Supernatant (S), wash (W), and pellet (P) fractions were then analyzed by Western blotting. (D) Wild-type and mutant versions of SpoIIDWch are equally expressed in E. coli. The indicated strains were treated as described for 600 of 1) were harvested at 6 h with or without addition of IPTG and resuspended in Laemmli buffer. The protein was then detected by Western blotting using polyclonal mouse anti-SpoIIDWch. Bands marked with stars are nonspecific due to background immunity of mice against E. coli. (E) Measurement of CFU per OD600 upon overexpression of SpoIIDs. The indicated strains were grown, and production of SpoIIDs was induced or not induced by the addition of IPTG. After 6 h of induction, OD600 levels were measured, serial dilutions were spread on LB plates, and colonies were counted. Download FIG S5, PDF file, 0.2 MB.(A and B) Copyright © 2019 Jacquier et al.2019Jacquier et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Wchin vivo, we overexpressed this protein in E. coli. Overexpression of SpoIIDWch in a wild-type E. coli strain, akin to overexpression of SpoIIDBsu, did not cause any growth defect or filamentation  in single mutants ΔdamX, ΔdedD, and ΔrlpA and in a triple mutant. IPTG-induced overexpression of SpoIIDs caused a growth defect in the triple mutant only , SpoOA domain , SspA domain , and DpaA domain  in a selection of bacterial, archeal, and eukaryotic genomes is shown here. The length of the colored bar represents the percentage of species among the indicated order coding for a SpoIID homologue. Download Copyright © 2019 Jacquier et al.2019Jacquier et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B. subtilis has been well studied and was shown previously to be a lytic transglycosylase required for PG remodeling during sporulation of this bacterium . Moreover, SpoIID could also be coimmunoprecipitated with the MreB membrane anchor RodZ. Chlamydial SpoIIDs show only a low level of amino acid identity with SpoIIDBsu (19.9% for W. chondrophila and 19.0% for C. trachomatis). Moreover, chlamydial SpoIIDs are quite divergent from each other, with a low identity level of 23.74% between SpoIIDWch and SpoIIDCtr  SpoIIDWch is expressed only upon induction by tetracycline (aTC). SpoIIDWch was detected by immunofluorescence using specific anti-SpoIIDWch antibodies (green), concanavalin A (red), and DAPI (blue). Bar, 40 μm. (B) Morphology of RBs upon overexpression of SpoIIDWch was observed using a C. trachomatis-specific anti-lipopolysaccharide (LPS) antibody (green). Bar, 1 μm. (C) Localization of SpoIIDWch in C. trachomatis RBs. Cells were treated as described for panel A, and pictures were taken with same magnification as was used for panel B. (D) Quantification of sizes of the RBs shown in panel B was performed using the ImageJ software, and the results were analyzed by the use of GraphPad Prism software. Download FIG S6, PDF file, 0.2 MB.Overexpression of SpoIIDCopyright © 2019 Jacquier et al.2019Jacquier et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Wch localizes at the division septum, we can hypothesize that chlamydial SpoIIDs are involved in PG degradation at the division septum and that this degradation is linked to inner membrane invagination and thus could be involved in septation. Interestingly, SpoIIDWch seems to degrade PG by two different mechanisms; we observed lytic transglycosylase activity on intact PG sacculi (resulting in anhydromuropeptides) and a muramidase activity on naked glycan chains (resulting mainly in nonanhydro glycan moieties). This potential bifunctional role of SpoIIDWch as lytic transglycosylase and muramidase, which is not shared by SpoIIDBsu, might thus be involved in PG degradation with amidases, which are present in Chlamydiales  supplemented with 10% fetal calf serum at 37°C and 5% CO2. Vero cells were harvested and counted. A cell suspension of 5 × 105 cells/ml was allowed to adhere overnight. Vero cells were then infected with a 2,000× dilution of W. chondrophila in DMEM supplemented with fetal calf serum. Infection was synchronized by centrifugation at 1,790 × g for 15 min and incubation at 37°C for an additional 15 min. The medium was then removed, and the cell layer was washed once with phosphate-buffered saline (PBS) and further incubated in fresh medium at 37°C in a 5% CO2 atmosphere.Vero cells (ATCC CCL-81) and McCoy (ATCC CRL-1696) cells were cultivated, and Vero cells were infected with eviously . Briefly18 column (Waters) . The mobile phase consisted of solvent A (0.1% formic acid–Milli-Q water) and solvent B (0.1% formic acid–acetonitrile). The gradient was set as follows: 0 to 3 min, 2% to 5% solvent B; 3 to 6 min, 5% to 6.8% solvent B; 6 to 7.5 min, 6.8% to 9% solvent B; 7.5 to 9 min, 9% to 14% solvent B; 9 to 11 min, 14% to 20% solvent B; 11 to 12 min, hold at 20% solvent B, keeping the constant flow of 0.250 μl/min; 12 to 12.1 min, 20% to 90% solvent B with a flow rate ramp of 0.250 μl/min to 0.300 μl/min, further holding the same flow rate and percentage of solvent B until 13.5 min; 13.5 to 13.6 min, 90% to 2% solvent B; 13.6 to 16 min, hold at 2% solvent B. The flow rate was shifted again to 0.250 μl/min at 16.1 min. Chromatographic column was equilibrated until 18 min for next analysis. Chromatographic data were recorded at 204 nm.PG was purified from the corresponding bacteria according to the protocol reported previously by de Jonge et al.  and as dm/z, with a scan time of 0.25 s. The instrument was operated in positive electrospray ionization mode. The capillary and sample cone voltages were 3 kV and 40 V, respectively. The gas flow rates were set at 100 and 500 liters/h for the cone gas and desolvation gas, respectively. The source temperature was 120°C, and the desolvation temperature was 350°C. For MSE, the low collision energy level was set at 6 eV, and the high collision energy level was ramped from 15 to 40 eV. A leucine-enkephalin reference was used at a concentration of 200 pg/ml as the lockmass, with a continuous flow rate of 5 μl/min and 0.25-s scan time, for an accurate analysis. All the processes involved in acquisition and analysis of data were controlled by Waters UNIFI software. Structural characterization of muropeptides was done on the basis of their MS and MS/MS fragmentation pattern data (Mass spectrometry was performed on a Xevo G2-XS QTOF (Waters), which is a quadrupole time of flight mass spectrometer. The scan range ran from 100 to 2,000 ern data .W. chondrophila were harvested 24 h p.i. and lysed by incubation in radioimmunoprecipitation assay (RIPA) buffer (Sigma) as described by the manufacturer. Lysates were clarified by centrifugation at 8,000 × g for 10 min at 4°C. Antibodies raised against RodZ of W. chondrophila . DNA was removed by selective digestion with DNase from an Ambion DNA-free kit . cDNA was then synthesized by reverse transcription using a Goscript reverse transcription system (Promega), and WchspoIID cDNA was quantified by qPCR performed on 4 μl of cDNA mixed with 10 μl of iTaq Universal SYBR green mix (Bio-Rad), 4.8 μl of water, and 0.6 μl each of primers SpoIID_RT_F and SpoIID_RT_R –10 mM Tris-HCl (pH 7.5) for 1 h at 60°C to remove Braun's lipoprotein. After addition of 1% (wt/vol) SDS, the reaction mixtures were heat inactivated and detergent was removed by washing in Milli-Q water. A 20-μl volume of purified PG was then incubated on ice with 2 μg of the indicated protein diluted in a total of 50 μl of PBS for 30 min. The pellet and supernatant were separated by ultracentrifugation. The pellet was washed once with PBS. Equivalent amounts of supernatant, wash, and pellet were separated by SDS-PAGE and detected by Western blotting –0.25 M NaOH, and incubated overnight at 37°C. The suspension was then neutralized with HCl, and excess dye was removed by repeated washes with PBS, to the point where the supernatant remained clear. A 40-μl volume of labeled PG was then incubated with 6 μg of the indicated proteins in a total of 100 μl PBS for 45 min at 37°C under conditions of agitation. PG was then sedimented by centrifugation at 16,000 × g for 10 min. The supernatant was collected, and its absorbance was measured at 595 nm using a Fluo Star Omega plate reader .The PG release assay was adapted from a method described previously by Morlot et al. . BrieflyE. coli strains were grown overnight in LB medium at 37°C. Optical density at 600 nm (OD600) was measured by spectrophotometry. All the strains were diluted to an OD600 of 0.2 per ml and incubated 1 h at 37°C. IPTG was then added at 1 mM if indicated. Growth was measured by spectrophotometry 2, 4, and 6 h after the dilution. Microscopy was performed 6 h after dilution, using a 100× objective on an Axioplan 2 microscope (Zeiss). Lengths of bacteria were measured using ImageJ, and a minimum of 100 bacteria were classified as normal or elongated in duplicate in two independent experiments.


Juvenile idiopathic arthritis (JIA) is an inflammatory arthritis of unknown etiology, which lasts for greater than 6 weeks with onset before 16 years of age. JIA is the most common chronic rheumatic disease in children. NSAIDs have been the mainstay of initial management with naproxen (NAP) being commonly used, but they may cause serious side effects such as gastric ulcers which can be reduced by concomitant administration of proton pump inhibitors, such as esomeprazole (ESO).Primary objective was to evaluate the safety and tolerability of 3 fixed doses of NAP/ESO in JIA patients aged 12 to 16 years. Forty-six children and adolescents with JIA by International League of Associations for Rheumatology criteria, mean age of 13.6 years, from 18 US sites were prospectively enrolled over 2 years and followed for up to 6 months. Doses of the NAP/ESO fixed combination were based on baseline weight. The exploratory efficacy outcome was assessed with the ACR Pediatric-30, − 50, − 70, − 90 Response and the Childhood Health Assessment Questionnaire (CHAQ) discomfort and functional scores at months 1, 3, and 6 as change from baseline. Occurrence and causality were assessed for treatment emergent AEs (TEAEs) and discontinuations were monitored monthly.Forty-six patients received at least 1 dose of naproxen/esomeprazole and 36 completed the trial. Thirty-seven (80.4%) had at least 1 treatment emergent adverse event (TEAE) and, with the exception of 2 events in one patient, all of the TEAEs were mild or moderate. Frequent TEAEs (≥5% of patients) were upper respiratory tract and gastrointestinal related. Eleven (23.9%) had at least 1 TEAE considered to be related to study drug. Four patients (8.7%) discontinued due to a TEAE with one of these being the only serious AE reported, acute hepatitis. Mean number of active joints at baseline was 3.1. Improvement in JIA signs and symptoms occurred at most assessments and by month 6, the percentage of patients with an ACR Pediatric-30, − 50, − 70, and − 90 Response was 47.1, 38.2, 32.4, and 17.6%, respectively. The percent of patients achieving ACR Pediatric response increased over time. CHAQ discomfort improved at each assessment and functional scores improved at all assessments for ‘Arising, Walking, and Activities’ with several improved for ‘Dressing and Grooming, Eating, Hygiene, and Grip’. There was no indication of a dose-related efficacy effect.NAP/ESO was well tolerated in JIA patients aged 12 to 16 years with high levels of response to ACR criteria. No new safety signals were identified for the well-characterized components of this fixed dosed JIA treatment, which was developed to reduce the risk of gastric ulcers.NCT01544114. Registered February 21, 2012.Clinicaltrials.gov, The online version of this article (10.1186/s12969-018-0260-y) contains supplementary material, which is available to authorized users. Juvenile idiopathic arthritis (JIA) is an inflammatory arthritis of unknown etiology, which lasts for greater than 6 weeks with onset before 16 years of age . Per theFor pediatric patients up to 17 years of age, a delayed-release form of esomeprazole magnesium is approved in many countries for the short-term treatment 8 weeks) of gastroesophageal reflux disease and has been shown to be safe and effective , where s was the standard deviation of the data on a natural log scale.For the PK analysis, summary statistics of trough naproxen plasma concentration (lowest plasma concentration from pre-dose to 3 h post-dose) during steady state were presented by dose group and visit. Empirical Bayes’ estimates of the individual PK parameters were generated based on the final structural and variance parameter estimates and the individual plasma concentration measurements using nonlinear mixed effects modeling (NONMEM). Esomeprazole PK parameter estimates derived from this Population (Pop) PK analysis were analyzed and listed by study dose group and individual patient. The geometric %CV was calculated as 100 × √ patients).Due to a theoretical risk of decreased levels for iron, vitamin B12, and magnesium during long-term PPI exposure, potential alterations in these moieties during 6 months of PPI therapy were evaluated and no significant changes were seen.0.75; V/F: 38.68 vs. 21.03 L/70 kg). Individual oral CL/F estimates in adolescent subjects (ranging from 3.84 to 131.91 L/h/70kg0.75) were within the range of estimates observed in adults (3.43–136.62 L/h/70kg0.75) [max and Tmax data in frequently sampled pediatric patients were comparable to those reported in pediatric studies [Forty patients supplied samples for esomeprazole and 41 provided samples for naproxen. Esomeprazole concentrations observed in adolescents are comparable to those previously reported in adult and adolescent studies . Geometr0kg0.75) . Total e0kg0.75) –17. Napr studies , 19 and  studies . Naproxe studies . See supThe percentage of patients achieving ACR Pediatric response increased over time Fig. .The mean baseline score for physician’s assessment of disease activity was 2.58 and individual mean improvement from baseline was − 0.67, − 0.99, and − 1.23 at Months 1, 3, and 6, respectively.Mean CHAQ global assessment of Well-Being baseline score was 3.43 and mean improvement from baseline was − 0.80, − 0.93, and − 1.39 at Months 1, 3, and 6.Mean CHAQ functional scores indicated improvement from baseline for 3 of the functional areas . For 4 of the functional areas , the mean change from baseline indicated an improvement or no change (mean change 0.0) at each assessment. For the remaining functional area of ‘reach’, there was no change at each post-baseline assessment except Month 1 (mean change 0.2).The mean baseline score for the CHAQ disability index was 0.506. The mean improvement from baseline was − 0.069, − 0.064, and − 0.155 at Months 1, 3, and 6, respectively.The mean baseline number of joints with active arthritis was 3.1. The individual mean change indicated an improvement from baseline to each post-baseline assessment. The mean improvement from baseline was − 1.1, − 0.4, and − 0.6 at Months 1, 3, and 6, respectively.The mean baseline number of joints with limited range of motion was 1.7. The individual mean improvement from baseline was − 0.9, − 0.1, and − 0.2 at Months 1, 3, and 6, respectively.The mean baseline serum CRP was 8.807 mg/mL with a mean change of 0.169, − 1.076, and − 1.120 at Months 1, 3, and 6, respectively, indicating individual mean improvement at Months 3 and 6.The mean baseline serum ESR was 10.4 mm/hr. The mean individual change indicated improvement from baseline to Month 3. The mean change from baseline was 0.4, − 0.1, and 0.8 at Months 1, 3, and 6, respectively.CHAQ discomfort improved at each assessment from a baseline mean of 4.41. The mean improvement from baseline was − 1.24, − 1.21, and − 2.20 at Months 1, 3, and 6.There was no indication of a dose-related efficacy effect in any of the outcomes.Adherence with study medication was good with 73.9% of patients (34 of 46) with ≥80% and ≤ 120% compliance and 21.7% (10 of 46 patients) demonstrating < 80% or > 120% (non-compliance). Two subjects were lost to follow-up and therefore did not have an overall compliance calculated.N = 21) displayed a greater increase in percentage of patients with ACR Response 30, − 50, − 70, and − 90 when compared to those using NAP/ESO as their primary therapy  of them receiving 6 months of study drug (defined as ≥166 days on study drug). The PK results approximated the esomeprazole PK data from previously published pediatric studies. The naproxen Cterature –20. In aThe fixed combination of NAP/ESO was well tolerated in JIA patients aged 12 to 16 years. No new safety signals were identified for the well-characterized components of this fixed dosed JIA treatment, which was developed to reduce the risk of gastric ulcers in patients requiring chronic naproxen therapy. Improvement in JIA signs and symptoms occurred at most assessments and by month 6, the percentage of patients with an ACR Pediatric-30, − 50, − 70, and − 90 Response was 47.1, 38.2, 32.4, and 17.6%, respectively.Additional file 1:Supplemental Data. (DOCX 54 kb)


Total N = 297) valued leadership potential more highly than leadership performance, but only for male candidates. By contrast, female candidates were preferred when they demonstrated leadership performance over leadership potential. The findings reveal an overlooked potential effect that exclusively benefits men and hinders women who pursue leadership positions that require leadership potential. Implications for the representation of women in leadership positions and directions for future research are discussed.Two experiments tested the value people attach to the leadership potential and leadership performance of female and male candidates for leadership positions in an organizational hiring simulation. In both experiments, participants ( Given that women now represent 40% of the global working population , it woulLeadership potential specifically refers to exhibiting the qualities that signal future leadership effectiveness  revealed that women were evaluated more favorably than men on overall job performance ratings. Yet women were rated lower than men on the measure of future performance and promotability . Previous research has found that assessments of women were higher than those of men on leadership performance but lower than those of men on vision and strategy e.g., . Differetability . Such ditability . Nonethetability .communal , whereas men are expected to be agentic  revealed a bias for men in male-dominated roles . However, that role congruity bias was attenuated when information clearly highlighted a candidate’s high competence. We hypothesized that a female candidate’s leadership potential may only be acknowledged if she is unambiguously a high performer .Although research indicates that evaluations of leaders and promotion to leadership positions are likely to be biased in favor of men, a meta-analysis . We expected that participants would prefer leadership potential more in male candidates than in female candidates (Hypothesis 2). More importantly, when it comes to candidate choice, we hypothesized that participants would prefer leadership potential over leadership performance in male candidates (Hypothesis 3); but leadership performance over leadership potential in female candidates (Hypothesis 4). In addition, we hypothesized that high leadership potential male candidates would be selected more than high potential female candidates (Hypothesis 5).All experiments were carried out in accordance with the recommendations of the School of Psychology Ethics Committee at the University of Kent, United Kingdom. The protocol was approved using the School of Psychology Ethics system. All participants gave written informed consent in accordance with the Declaration of Helsinki. The research was conducted in accordance with guidelines from the University of Kent Research Ethics (Human Participants) Committee, the Economic and Social Research Council (ESRC) Research Ethics Framework, and the ethical guidelines from the British Psychological Society (BPS).Mage = 36.38, 79.6% employed) via Amazon MTurk. The quasi-experimental design was a 2  × 2  × 2  mixed design, with leadership characteristic and candidate gender as within-participant factors. All additional candidate information  was counterbalanced.We recruited 98 participants  to a number of different industries. Imagine that you work in a human resources role and you are part of the team responsible for recruiting and hiring new employees. ALPHATech are currently expanding their business and as part of this are recruiting for a number of positions within the company. Imagine that you are part of the hiring panel and you have been given some candidates to evaluate.”“Candidate potential and performance were manipulated by adjusting the score on two assessments: leadership achievement and leadership potential. Specifically, as in “The LAI gauges leadership achievement, defined as an individual’s observed  leadership performance at the current stage in his or her career. An achievement score of 83 places this applicant in the top 17% of people who have been assessed [An achievement score of 96 places this applicant in the top 4% of people who have been assessed].1, which varied depending on the condition :The Assessment of Leadership Potential score was accompanied by the following paragraph“The ALP gauges leadership potential, defined as the employee’s predicted leadership performance in the near future. A score of 96 indicates that this applicant predicted future leadership performance is estimated to be in the top 4% of people who have been assessed [A score of 83 indicates that this applicant predicted future leadership performance is estimated to be in the top 17% of people who have been assessed].”Thus, in the leadership potential condition, the applicant had received a higher score on potential (top 4%) and a more moderate score (top 17%) on leadership achievement, whereas in the leadership performance condition, the applicant had received a moderate leadership potential score (top 17%) and a high performance score (top 4%). High and moderate scores were used rather than high and low scores, in order to focus attention on the dimension at which the candidate excelled rather than suggesting any weakness see . The focThis candidate has great prospects. She has some exciting new ideas for the future of the team and the organization, which could offer the opportunity to increase sales and performance in the future.” [Leadership Potential]“The applicant is highly capable, and has consistently performed above his own objectives and that of the organizations. The performance in his current role has exceeded expectations.” [Leadership Performance]“Candidate hiring was measured using two items on a 9-point rating scale (α = 0.78): “How interested would you be in hiring each applicant?,” “To what extent do you think hiring each applicant would be a good decision or a bad one?” Lower values indicate less hiring intention.not at all successful, 9 – very successful).Expected success was measured using one item asking participants “How successful do you think each applicant will be in their career?” (1 – first (most impressive) to fourth (least impressive).2Résumé evaluation was measured by asking participants to compare all four applicants and decide “in your opinion, which applicant has the most impressive résumé?” They were required to rank candidates from first) to worst future performance (fourth).Future performance was measured with an order of preference based on performance, “which applicant do you think will perform better by the fifth year at ALPHATech?” Candidates were ranked from best future performance  × Candidate Gender  × Participant Gender  mixed ANOVA to analyze the evaluation items of candidate hiring and expected success. We hypothesized that participants would be more willing to hire candidates with leadership potential and would expect those candidates to be more successful than candidates with leadership performance (Hypothesis 1). Furthermore, we expected these effects to be stronger for male candidates (Hypothesis 2 and Hypothesis 3). We did not hypothesize participant gender effects but included this factor as exploratory.Friedman tests and Wilcoxon Signed Ranks tests were used to analyze whether there were differences in the choice-based rankings of each candidate’s résumé and expected future performance. We expected participants to rank the male candidate with leadership potential higher than the male candidate with leadership performance on the evaluation of résumés and expected future performance. We expected the opposite pattern for female candidates (Hypothesis 4). Finally, we expected participants to rank the male candidate with leadership potential higher than the female candidate with leadership potential in both the evaluation of résumés and expected future performance (Hypothesis 5).F = 5.15, p = 0.025, η2 = 0.05, with female candidates rated as more likely to be a good hire than male candidates, see F = 1.40, p = 0.240, η2 = 0.01, as was the main effect of participant gender, F = 0.42, p = 0.838, η2 < 0.001. There was a significant Candidate Gender × Participant Gender interaction, F = 9.77, p = 0.002, η2 = 0.09. Simple main effects of candidate gender within levels of participant gender were analyzed. There was a significant difference for female participants’ evaluation of male and female candidates, F = 12.09, p = 0.001, η2 = 0.11, who expressed a preference for female candidates over male candidates overall  = 0.46, p = 0.499, η2 = 0.01. There were no simple main effects of participant gender within level of candidate gender. Female and male participants did not make significantly different hiring evaluations of female candidates, F = 3.59, p = 0.06, η2 = 0.04, or of male candidates, F = 1.88, p = 0.174, η2 = 0.02. There was no significant Leadership Characteristic × Candidate Gender × Participant Gender interaction effect, F = 1.69, p = 0.196, η2 = 0.017.There was a significant main effect of candidate gender,  overall . This waF = 1.27, p = 0.263, η2 = 0.01, participant gender F = 2.34, p = 0.129, η2 = 0.02, or leadership characteristic F = 2.57, p = 0.112, η2 = 0.03 (which does not support Hypothesis 1). There Candidate Gender × Participant Gender interaction effect was not significant, F = 3.10, p = 0.082, η2 = 0.03, and a non-significant Leadership Characteristic × Participant Gender interaction, F < 0.001, p = 0.995, η2 < 0.01. There was a significant Leadership Characteristic × Candidate Gender interaction, F = 4.28, p = 0.041, η2 = 0.04. Consistent with Hypothesis 2, there was a preference for leadership potential over leadership performance for male candidates only, F = 5.12, p = 0.026, η2 = 0.05, see F = 0.001, p = 0.981, η2 < 0.001. There was no significant differentiation between male and female leadership potential candidates, F = 0.52, p = 0.473, η2 = 0.05, or between male and female leadership performance candidates, F = 3.56, p = 0.06, η2 = 0.04.There were no significant main effects of candidate gender, F = 5.85, p = 0.017, η2 = 0.06. We decomposed the three-way interaction by participant gender. Simple interactions showed that the Leadership Characteristic × Candidate Gender interaction was only significant among female participants, F = 8.37, p = 0.005, η2 = 0.08, not among male participants, F = 0.08, p = 0.783, η2 < 0.01.Our exploratory analysis for participant gender revealed a significant Leadership Characteristic × Candidate Gender × Participant Gender interaction, F = 7.94, p = 0.006, η2 = 0.08. F = 5.32, p = 0.023, η2 = 0.05. Female participants did not differentiate significantly between female candidates based on leadership characteristic, F = 1.15, p = 0.287, η2 = 0.01, or between male and female candidates with leadership potential, F = 0.54, p = 0.465, η2 < 0.01.The second order simple effect was significant among female participants who differentiated between candidates with leadership performance, 2(3) = 88.51, p < 0.001, see Z = −6.36, p < 0.001 (Hypothesis 3). In contrast, female candidates with leadership performance were ranked higher than female candidates with leadership potential, Z = −4.70, p < 0.001 (Hypothesis 4). Furthermore, male candidates with leadership potential were ranked higher than female candidates with leadership potential, Z = −6.27, p < 0.001 (Hypothesis 5). Moreover, female candidates with leadership performance were ranked higher than male candidates with leadership performance, Z = −5.92, p < 0.001. In brief, in support of our hypotheses, male candidates with leadership potential were ranked as more impressive than male candidates with leadership performance. In contrast, female candidates with leadership performance were ranked as more impressive than female candidates with leadership potential.3A Friedman test showed that the ranking evaluations of each candidate résumé were different, χ2(3) = 78.59, p < 0.001, see Z = −6.12, p < 0.001 (Hypothesis 3). In contrast, female candidates with leadership performance were ranked higher than those with leadership potential, Z = −4.65, p < 0.001 (Hypothesis 4). Furthermore, male candidates with leadership potential were ranked higher than female candidates with leadership potential, Z = −6.00, p < 0.001 (Hypothesis 5). Finally, female candidates with leadership performance were ranked higher than male candidates with leadership performance, Z = −5.93, p < 0.001. In brief, results supported our hypotheses, with male candidates with leadership potential ranked more highly than those with leadership performance, but that this would not be the case for female candidates. Indeed, female candidates with leadership performance were ranked higher than female candidates with leadership potential.A Friedman test showed that the rankings reflecting expectations of each candidate’s future performance were different, χExperiment 1 provides the first experimental evidence that female and male candidates’ leadership potential and leadership performance are evaluated differently. We did not find evidence for Hypothesis 1, an overall preference for potential. In line with an overlooked potential pattern, we found that participants expected male candidates with leadership potential to be more successful than male candidates with leadership performance (Hypothesis 3), although this was not the case for the candidate hiring measure. When participants ranked female candidates, they preferred leadership performance over leadership potential consistently across measures (support for Hypothesis 4). Interestingly, when participants were asked to rank candidates in evaluation of résumés and on future performance, female candidates’ leadership performance was preferred over that of male candidates. This type of ranking decision closely matches actual hiring processes, where final choices rely on rule-based selection criteria .We did not hypothesize effects of participant gender, but exploratory analysis revealed some differences. Specifically, a three-way interaction on candidates’ expected success showed that the two-way interaction was only significant among female participants. When judging candidates’ expected success, female participants rated female candidates with leadership performance as likely to be more successful than male candidates with leadership performance. Female participants also expected male candidates with leadership potential to be more successful than male candidates with leadership performance.In this study, female candidates were rated as more hirable than male candidates. This unexpected finding is in line with a recent meta-analysis which showed that women are rated more effective than men in senior levels . The stiN = 199; 126 females, 73 male Mage = 35.02, 78.4% in full or part-time employment) were recruited via an international online database, Amazon MTurk. The quasi experiment was a Leadership Characteristic × Candidate Gender × Participant Gender mixed design, with leadership characteristic and candidate gender as within-participant factors. All participants were exposed to a total of four résumés manipulating leadership characteristic  and candidate gender . To ensure consistency in other relevant résumé information, participants randomly received counterbalanced combinations of additional background information for each candidate.Participants . The background information and leadership scores  were the same as shown in Experiment 1. In Experiment 2, the résumés were made relevant to the hiring of a more senior candidate by changing candidates’ previous work experience to include at least one well known tech or communications company and by providing reviews from other people (previous employer and Tell Inc. CEO) and self-descriptions by the candidate. These comments reinforced either the candidates’ future leadership potential or previous leadership performance. The following examples show quotes from a CEO about a female candidate with leadership performance and about a male candidate with leadership potential, respectively:“Christine is clearly a candidate who has performed very highly throughout her career. She has shown from her past achievements and accomplishments that she is highly capable of performing to the highest standard. Christine is certainly at the top of her group in her professional achievements.”“Rupert is clearly a candidate who has shown excellent potential throughout his career. You can see from his budding talent and promise that he is highly capable of being one of the best in his field. Rupert is absolutely at the top of his vocation in terms of his professional potential.”Participants then completed the evaluative rating measures , immediately after reviewing each candidate. Next, all four résumés were presented simultaneously, so that participants could refresh their memory, and to minimize availability bias toward the most recently reviewed résumé. Participants then completed the dependent measures.strongly disagree) to 9 (strongly agree).Candidate hiring was measured using two items (α = 0.85): “I would hire this candidate” and “this candidate would be a good appointment.” Items were measured on a rating scale and ranged from 1 (strongly disagree) to 9 (strongly agree) , with first the most impressive.4Résumé evaluation was indicated by a choice of candidates, participants were asked “in your opinion, which applicant has the most impressive résumé?”, with first most likely to perform best.Future performance was assessed with the rank of candidates in response to the question “which candidate do you think will perform better by the fifth year?”  × Candidate Gender  × Participant Gender  mixed ANOVA was used to analyze the measures of candidate hiring and expected success. As in Experiment 1, we hypothesized that participants would be more likely to hire candidates with leadership potential and would expect those candidates to be more successful than candidates with leadership performance (Hypothesis 1). Furthermore, we anticipated that these effects should be stronger for male candidates (Hypothesis 2). We did not hypothesize participant gender effects but included this factor as exploratory.Friedman tests and Wilcoxon Signed Ranks tests were used to analyze whether there were differences in the choice-based rankings reflecting evaluations of each candidate’s résumé, future performance, and hire choice. Specifically, we expected participants to rank the male candidate with leadership potential higher the than male candidate with leadership performance on the evaluation of their résumés, future performance, and hire choice (Hypothesis 3). We predicted the opposite pattern for female candidates (Hypothesis 4). Finally, we expected participants to rank the male candidate with leadership potential higher than the female candidate with leadership potential in all ranking measures (Hypothesis 5).F = 15.05, p < 0.001, η2 = 0.07. Participants rated candidates who exhibited leadership performance on their résumé more favorably than candidates who displayed leadership potential  = 3.80, p = 0.053, η2 = 0.02. F = 3.14, p = 0.078, η2 = 0.02. All remaining effects were not significant  = 17.72, p < 0.001, η2 = 0.08. Candidates with leadership performance were rated as more likely to be successful than those with leadership potential  = 3.79, p = 0.053, η2 = 0.02. We decomposed the three-way interaction by participant gender. Simple interaction effects showed that the Leadership Characteristic × Candidate Gender interaction was only significant for female participants, F = 6.08, p = 0.015, η2 = 0.03, and not for male participants, F = 0.32, p = 0.571, η2 = 0.002. Second order simple effects show that female participants expected the female candidate with leadership performance to be more successful than the female candidate with leadership potential, F = 12.15, p = 0.001, η2 = 0.06. In addition, F = 9.12, p = 0.003, η2 = 0.04. Female participants did not differentiate significantly between the male candidates based on leadership characteristic, F = 0.04, p = 0.842, η2 < 0.001, or between male and female candidates with leadership performance, F = 0.15, p = 0.703, η2 = 0.001.There was a near significant Leadership Characteristic × Candidate Gender × Participant Gender interaction, 2(3) = 185.25, p < 0.001. Wilcoxon signed rank tests supported our hypotheses, and Z = −9.79, p < 0.001 (Hypothesis 3). In contrast, female candidates with leadership performance were ranked more highly than female candidates with leadership potential, Z = −6.19, p < 0.001 (Hypothesis 4). Furthermore, male candidates with leadership potential were ranked more highly than female candidates with leadership potential, Z = −9.76, p < 0.001 (Hypothesis 5). Finally, female candidates with leadership performance were ranked more highly than male candidates with leadership performance, Z = −7.61, p < 0.001.5A Friedman test showed that the rankings of the résumés differed, χ2(3) = 133.85, p < 0.001. Wilcoxon signed rank tests supported our hypotheses, and Z = −8.71, p < 0.001 (Hypothesis 3). In contrast, the future performance of female candidates with leadership performance was ranked more highly than that of female candidates with leadership potential, Z = −3.80, p < 0.001 (Hypothesis 4). Furthermore, male candidates with leadership potential were ranked more highly than female candidates with leadership potential, Z = −7.65, p < 0.001 (Hypothesis 5). Finally, female candidates with leadership performance were ranked more highly than male candidates with leadership performance, Z = −8.05, p < 0.001.A Friedman test showed that the four candidates’ future performances were ranked differently, χ2(3) = 164.84, p < 0.001. Wilcoxon signed rank tests supported our hypotheses, and Z = −9.56, p < 0.001 (Hypothesis 3). In contrast, female candidates with leadership performance were more likely to be the hire than those with leadership potential, Z = −4.36, p < 0.001 (Hypothesis 4). Furthermore, male candidates with leadership potential were more likely to be the hire than female candidates with leadership potential, Z = −8.44, p < 0.001 (Hypothesis 5). Finally, female candidates with leadership performance were more likely to be the hire than male candidates with leadership performance, Z = −8.42, p < 0.001.A Friedman test showed that hiring preference differed among the four candidates, χExperiment 2 provides evidence that candidates’ gender moderates evaluations of their leadership characteristics. Consistent findings across the ranking measures provide clear evidence regarding the overlooked potential effect. We found that when participants ranked male candidates there was a preference for potential (Hypothesis 3), whereas leadership potential was overlooked when they ranked female candidates (Hypotheses 4 and 5). Indeed, consistent with Experiment 1, when participants judged female candidates, leadership performance was preferred over leadership potential. Moreover, the finding that leadership potential led to an upgrading of  male candidates relative to female candidates, and that leadership performance led to a downgrading of male relative to female candidates seems highly consistent with the interpretation that gender role expectations moderated judgments of the candidates.In our exploratory analysis we also found some evidence that participant gender affected these judgments. Specifically, an interaction between candidate gender and leadership characteristic on expectations about candidates’ success was significant among female participants but not male participants. Female participants rated the male candidate with leadership potential higher than the female candidate with leadership potential. Additionally, female participants expected the female candidate with leadership performance to be more successful than the female candidate with leadership potential.Our findings provide several new empirical and theoretical contributions. Overall, these studies provide the first experimental evidence that a candidate’s gender can affect evaluators’ assessment of the value of their leadership potential and leadership performance. In both experiments, consistent with our Hypotheses 3, 4, and 5, leadership potential was preferred when participants ranked male candidates, whereas potential was overlooked when participants ranked female candidates. Male candidates that demonstrated higher potential were perceived to have a more impressive résumé and were expected to perform better in the future than male candidates who demonstrated higher performance. In contrast, female candidates who demonstrated higher performance were perceived to have a more impressive résumé and were expected to perform better than female candidates who demonstrated higher potential. If these findings were extrapolated to real hiring situations, they would mean that whilst women’s past performance would have to at least as good as men’s, women would be held to higher standards in selection processes because their leadership potential would be less likely to be recognized than men’s. The findings emerged most clearly when participants ranked rather than rated candidates. The ranking data are likely to have higher ecological validity given that most recruitment procedures conclude with a ranking process in order to decide which candidate to hire.Why might men with future leadership potential have a distinctive advantage? One explanation can be drawn from role incongruity theory which highlights that people have a powerful implicit association between leadership and agentic traits . Female We explored the impact of gender on preference for leadership potential and/or leadership performance. On candidate choice rankings an unexpected but consistent finding was that participants prioritized female’s leadership performance over that of male candidates. It could be that women are implicitly required to show greater evidence of competence to overcome stereotypically negative performance expectations, particularly in male gender-typed job domains . TherefoDespite generally consistent findings, a few inconsistencies merit discussion. These may reflect that the two studies assessed judgments relating to different levels of seniority (higher in Experiment 2). In Experiment 1, but not in Experiment 2, participants perceived female candidates to be a better hire overall. This unexpected result might have been driven by participants’ reactions to encountering counterstereotypical high-performing women, an advantage that may be worth exploring in case it is limited to judgments about junior leadership roles.There was also some evidence of gender ingroup bias in both experiments but it was not ubiquitous. Although ingroup bias is a robust social psychological phenomenon , particuGoing beyond previous research, these studies demonstrated that when faced with a choice, judges consistently ranked male candidates with leadership potential over their female counterparts. Our ranking findings are of particular significance as they mirror the majority of selection and recruitment decisions, whereby only one candidate can be offered the job. Moreover, processes that identify and fast-track leadership potential are already in place in many organizations e.g., . UnderstWe have provided evidence that leadership potential and leadership performance can yield different hiring and evaluation outcomes for men and women. Various limitations need to be considered before making strong conclusions. First, the extant literature lacks a well-developed empirical foundation for the theoretical distinction between leadership potential and leadership performance. We therefore relied on a general definition of leadership potential, which might not fully encompass the entire array of traits linked to leadership potential or their relationship with leadership performance. We chose to focus distinctly on either past performance or future potential to avoid confusion. We gave the manipulations context and reinforcement in Experiment 2 by providing a richer view of the candidate . Overall, even if a more comprehensive basis for the distinction could improve the design of descriptions in vignettes, the results do show that participants responded differently to the leadership potential and leadership performance depictions of candidates.We used a crowdsourcing platform which had the advantage of using a real-world sample of employed people across a range of occupations. Nonetheless, it is possible that this approach also introduced more unexplained variability in the sample  than might be attained with a more homogenous sample . Future research could investigate the generality of the overlooked potential effect in single organizational contexts, or compare different organizational contexts that are more typically male- or female-dominated. Moreover, it is conceivable that differences amongst participants’ own occupancy of leadership positions, may have influenced their responses. Future research should investigate potential moderating effects of participant leadership experience. A further way to pursue future studies would be to test the effect using samples of hiring managers and members of promotion panels.Additional directions for research include investigating boundary conditions for the effect such as different leadership goals , or culture. For example, high potential women are regarded as having higher diversity value . It woulThe degree of role incongruity could also be pursued as a moderating factor. A further subtlety may be that the linguistic framing of the role positions may affect whether or not potential is overlooked. For example, Real hiring decisions are based on choices, which our ranking measures simulated. However, the decision to use ranking measures imposed limits to our capacity to investigate moderation effects. Moreover, hiring decisions are often based not only on résumé evaluations but also subsequent rounds of interviews. The present research only speaks to the first stage of this selection process. It may be that these interviews either ameliorate or exacerbate the overlooked potential effect, which also warrants investigation in future research.The present research has practical implications for organizations, and possibly even beyond. For example, if preference for leadership potential in men is a generic phenomenon, it may well confer unfair advantages well beyond commercial and business contexts . For any organization, ensuring that hiring processes are fair and offer equal opportunities is fundamental for attaining gender equity in leadership positions. Given that employers typically regard leadership potential as a desirable trait , raisingAll experiments were carried out in accordance with the recommendations of the School of Psychology Ethics Committee at the University of Kent, United Kingdom. The protocol was approved by the School of Psychology Ethics process. All participants gave written informed consent in accordance with the Declaration of Helsinki. The research was conducted in accordance with guidelines from the University of Kent Research Ethics (Human Participants) Committee, the Economic and Social Research Council (ESRC) Research Ethics Framework, and the ethical guidelines from the British Psychology Society (BPS).AP and GR conceived of the presented idea and took the joint lead in writing the manuscript. AL, DA, and FT contributed in theory development. AL and DA bolstered analytical methods. GR and DA encouraged AP to pursue the application of leadership potential and gender to leadership selection and supervised the findings of this research. DA worked on revisions for the final version. All authors provided critical feedback and helped to shape the overall research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.