Pseudomonas aeruginosa, an opportunistic human pathogen, is very complex. In the PAO1 reference strain, about 10% of genes encode transcriptional regulators, many of which have undefined regulons and unknown functions. The aim of this study is the characterization of PA2577 protein, a representative of the Lrp/AsnC family of transcriptional regulators. This family encompasses proteins involved in the amino acid metabolism, regulation of transport processes or cell morphogenesis. The transcriptome profiling of P. aeruginosa cells with mild PA2577 overproduction revealed a decreased expression of the PA2576 gene oriented divergently to PA2577 and encoding an EamA-like transporter. A gene expression analysis showed a higher mRNA level of PA2576 in P. aeruginosa ΔPA2577, indicating that PA2577 acts as a repressor. Concomitantly, ChIP-seq and EMSA assays confirmed strong interactions of PA2577 with the PA2577/PA2576 intergenic region. Additionally, phenotype microarray analyses indicated an impaired metabolism of ΔPA2576 and ΔPA2577 mutants in the presence of polymyxin B, which suggests disturbances of membrane functions in these mutants. We show that PA2576 interacts with two proteins, PA5006 and PA3694, with a predicted role in lipopolysaccharide (LPS) and membrane biogenesis. Overall, our results indicate that PA2577 acts as a repressor of the PA2576 gene coding for the EamA-like transporter and may play a role in the modulation of the cellular response to stress conditions, including antimicrobial peptides, e.g., polymyxin B.The regulatory network of gene expression in  Escherichia coli and its paralogue—the AsnC protein. Lrp is one of the best-studied bacterial transcriptional regulators which acts as a global factor, considered to control the expression of almost 30% of all E. coli genes . A. APA2577ruginosa B. The Swruginosa B. The prruginosa B.PA2577 was cloned into two pairs of BACTH vectors, derivatives of pairs pUT18C/pKT25 (pLKB4 and pLKB2) and pUT18/pKNT25 (pKGB4 and pKGB5), facilitating the linkage of tested protein with two Bordetella pertussis adenylate cyclase (CyaA) fragments (T18 and T25), either at the N or C-terminus of the protein . The reconstitution of CyaA activity was evaluated by a change in the color of the colonies on the MacConkey plates  and by measurements of β-galactosidase activity in the liquid medium. E. coli BTH101 cyaA cells transformed with pairs of BACTH vectors (one producing PA2577 fused with subunit T25 and the second with T18) showed very strong PA2577–PA2577 interactions  system  was used protein . The recractions C. The hi6 and glutaraldehyde cross-linking confirmed that the purified PA2577 has strong multimerization abilities. Protein dimers as well as higher-order oligomers, possibly octamers, have been observed  of the protein. The PA2577-His6 dimer has molecular weight of about 36 kDa, yet on the membrane, the bands around 40 kDa and 45 kDa were detected. Concomitantly, bands above 140–260 kDa, which may correspond to predicted octamers or even higher-order complexes, were observed. This could be explained by the differential protein solvation by SDS or the hydrophobicity of the protein. The other aspect is the possible presence of post-translational modification(s) or a potential ligand bound to PA2577 and co-purified with the protein upon cross-linking, which could have influenced the MW of PA2577 and formed higher-order complexes. PA2577 seemed to be very potent to multimerization and apart from the dimeric, tetrameric  or octameric form, it could create intermediate forms likely seen as smear or double/multiple bands on the membrane.Concomitantly, the analysis of the PA2577 oligomerization state using purified PA2577-Hisobserved D. As preOverall, the presented results indicated that PA2577 created higher-order complexes, which is a common property of other Lrp-like proteins ,18,38.P. aeruginosa, a PAO1161 ΔPA2577 strain was constructed and used in several phenotypic analyses ; however, no significant differences in comparison with the parental strain could be detected ) or the pMEB12 vector with PA2577 under the control of the arabinose-induced promoter (araBADp-PA2577 (PA2577+)) were cultured in a medium containing different concentrations of arabinose. This analysis showed a strong negative effect of PA2577 excess on P. aeruginosa growth (PA2577 expression by a very low concentration of arabinose (0.02%) did not have a significant effect on the kinetics of culture growth, whereas using higher concentrations of arabinose resulted in a more pronounced slowdown of bacterial growth (E. coli showed no significant differences between EV and PA2577+ cells upon growth in a medium containing various arabinose concentrations (data not shown), indicating that the effect of PA2577 excess on the slowdown of bacterial growth is specific for P. aeruginosa.To check how the overproduction of PA2577 influenced the bacterial growth, strains carrying pKGB8 ) as well as sole PA2578 ) overexpression was tested. In conditions when the whole PA2577–PA2578 operon was overexpressed, the negative effect on the growth of cells was even stronger than in the case of the sole PA2577 gene overexpression . The classification of 171 loci with an altered expression was performed according to PseudoCAP functional categories [PA0807, PA1698, PA2576 and PA3037) from the list of genes with an altered expression in response to the PA2577 excess were validated using an independent RT-qPCR analysis (PA1698 and PA2576) or increased (PA0807 and PA3037) mRNA level when comparing PA2577+ with EV+ cells.To identify genes with a changed expression in response to a mild PA2577 overproduction . Upregulation was observed for 39 genes coding for predicted bacteriophage proteins . The mos protein . This prase gene ,41. Upretathione .PA5160.1 tRNA gene was the most downregulated locus (FC = 0.2). Among repressed genes, two significantly downregulated loci attracted special attention—PA2664 and PA2576 . PA2664 encodes flavohemoprotein engaged in the oxidation–reduction process. Flavohemoglobins are poorly understood, but flavoHB from E. coli is involved in the repair of the lipid membrane caused by oxidative/nitrosative stress [PA2576 gene is also connected with membrane functions. It is oriented divergently to the PA2577 gene . The e stress . The dow577 gene A and encPA2576 is indeed the direct target of PA2577 and identify other potential PA2577 binding sites in the P. aeruginosa genome, a ChIP-seq analysis was performed. P. aeruginosa ΔPA2577 cells carrying pMEB84 (QlacI-tacp-PA2577-flag) were grown under selection in L broth with 0.05 mM IPTG and ΔPA2577 with pABB28.1 (QlacI-tacp-flag) grown under the same conditions being used as a negative control.To check if The sequencing of DNA immunoprecipitated using anti-flag antibodies confronted with a negative control revealed as much as 812 binding sites of PA2577. The vast majority of sites was characterized by a very low enrichment and a 2.5-fold enrichment was chosen as a cut-off value, eliminating about 97% of peaks . Such fiPA0600 and PA0641 (peak 1), PA0980 and PA0975 (peak 14), PA4334 and PA4342 (peak 18) and PA2581 and PA2573 (peak 25) genes from the PAO1 genome (Only four of obtained peaks had sharp shapes with a clear summit and they encompassed regions between 1 genome .PA0600–PA0641), with the summit of the peak identified in the PA0633 gene encoding a hypothetical protein related to phage functions, such as other genes from the cluster. The induction of the expression of most of the genes from the PA0612–PA0648 cluster was detected in the RNA-seq analysis in response to a mild PA2577 excess (prtN (PA0610) and ptrB (PA0612) encoding transcriptional regulators involved in stress response. Peak no. 14 was 3241 bp long and was the shortest one among 25 peaks identified for PA2577. In this region, the PA0980–PA0975 genes were located with the PA0978 encoding conserved hypothetical protein with a putative HTH-like and integrase/transposase catalytic domain, in which the peak summit was detected. However, no changes in gene expression in this region were observed under tested conditions in the performed RNA-seq analysis. Peak no. 18 was 7398 bp long and encompassed the PA4334–PA4342 genes, with a summit identified in the PA4336 gene. No changes in gene expression in this region were also observed.Peak no. 1 was 37545 bp long and encompassed the region encoding more than 40 genes .For three PA2577 peaks, the summits were located in intergenic regions  with the highest fold enrichment detected for peak no. 25 encompassing the r region . For peaanalysis . Peak no1–PA3031 . The sumanalysis . HoweverPA2576/PA2577 intergenic region  or in minimal medium (M9)  E.PA2577 and PA2576 was examined at the early (OD600~0.6) and late exponential (OD600~1.5) phases of growth of P. aeruginosa WT cultures. Results of the RT-qPCR analysis revealed that PA2577 expression was about five times higher in cells collected from the late exponential phase, whereas the PA2576 gene was expressed at a very low level independently of the phase of growth (about 10 times lower in comparison with PA2577)  F. Such aPA2576 gene encodes the protein predicted to be localized in the inner membrane and possesses two EamA-like domains, each containing five transmembrane helices. To determine the function of the PA2576 protein, a PAO1161 mutant lacking the PA2576 gene was constructed. Akin to ΔPA2577 strains, no phenotype changes were observed for the ΔPA2576 mutant in standard tests ) and pAMB9.37 (QlacI-tacp (EV)) in medium with IPTG used as the inducer revealed that the overproduction of PA2576 also strongly inhibited P. aeruginosa growth  was identified in one clone. The subsequent cloning of a full-length PA3694 and PA5006 into the BACTH vectors confirmed strong interactions between PA5006 and PA2576 and medium-strength interactions between PA3694 and PA2576 . Based on the amino acid sequence, it is predicted to be a lipoprotein [PA3694 is an uncharacterized small protein probably being a part of an operon of unknown function consisting of six genes (oprotein .PA5006 is the first gene from an operon consisting of seven loci (PA5006–PA5012) [PA5006 is the only putative gene in the cluster and probably the same as three other genes (waaP (PA5009), wapP (PA5008) and wapQ (PA5007)) belonging to the operon, encoding LPS kinase, which is responsible for the addition of phosphates to the inner core oligosaccharides of P. aeruginosa LPS  creating the operon encoding proteins responsible for the construction of the P. aeruginosa LPS core, which is built of highly phosphorylated sugars [–PA5012) . PA5006 nosa LPS . The foud sugars ,49. LPS d sugars .PA2576 and ΔPA2577 mutants in comparison with the WT strain were performed , but no significant differences could be observed in such conditions (data not shown). A phenotype microarrays analysis conducted on plate PM12B indicated an impaired metabolism of the ΔPA2576 mutant in the presence of polymyxin B under the highest tested concentrations, which suggested disturbances of membrane functions likely connected with LPS synthesis  for selected conditions for the Δynthesis A.The phenotype was confirmed by a standard growth experiment where PA2576- or PA2577-deficient cells grew significantly slower than the WT strain in the minimal M9 medium supplemented with citrate as the carbon source, polymyxin B and EDTA B.Overall, presented data indicated that PA2577 and PA2576 were involved in the control of membrane functions, which might be perturbed in the presence of agents influencing membrane integrity, e.g., polymyxin B.P. aeruginosa was described. Representatives of the Lrp/AsnC family of regulators appeared to be widely distributed among bacteria and archaea, whereas no Lrp-like TRs were identified in eukaryotic genomes so far [E. coli with three these types of regulators , but several bacteria contained dozens of such paralogues [P. aeruginosa, known for its high adaptability, nine Lrp/AsnC-type regulators could be identified .In this work, the characterization of the Lrp/AsnC transcriptional regulator PA2577 from s so far . Some prralogues . DiffereP. aeruginosa. An in silico analysis predicted a two-domain structure of PA2577, typical for FFRP, with the N-terminus involved in DNA binding and C-terminal ligand binding domain [Sulfolobus tokodaii [Pyrococcus sp. OT3 [Our work showed that PA2577 could form multimers, bind DNA and modulate gene expression in g domain . The postokodaii  or DM1 f sp. OT3 . In fact sp. OT3 ,50 or de sp. OT3 .PA2577 showed a 27% identity and 49% similarity with Grp, and conserved amino acids involved in interactions with glutamine in Grp seemed to also be present in PA2577, e.g., S31, Y62, D100, T127 and T129 . In the The formation of oligomeric structures by Lrp/AsnC proteins played a pivotal role in their interactions with DNA, sometimes allowing drastic conformation changes of the DNA duplex, such as DNA bending/wrapping ,53,54,55PA2576. Under tested conditions, PA2577 acted as the repressor of the PA2576 gene, transcribed divergently to PA2577.The performed RNA-seq analysis showed changes in the expression of 171 genes in response to PA2577. We did not observe a clear correlation of identified PA2577 binding sites in ChIP-seq  and genes that responded to PA2577 in the RNA-seq analysis under tested conditions, except for PA2576/PA2577 genes consisted of tracts of T and A bases, which may have helped in the modulation of the promoter structure by the bound PA2577 oligomer, possibly octamer, according to the model structure, and possible DNA bending and wrapping similar to LrpC from B. subtilis [PA2576p as well as PA2577p  are usually present in the target gene promoters [The intergenic region of subtilis . The AAA PA2577p B. Additi protein , e.g., tromoters . This maampDh3 or the PA3035–PA3037 operon. Intriguing was the observed decrease in the tRNA gene expression or upregulation of genes related to phages in response to PA2577 overproduction , even though the concentration of inducer was low. The induction of the expression of the PA0612–PA0648 gene cluster is often observed under different stress-generating growth conditions [Based on transcriptomic profiling, PA2577 might be indirectly involved in the control of several genes, e.g., oduction . The mecnditions ,39,61. AptrB encoding protein known as a repressor of the type III secretion system (T3SS) under the stress of DNA damage; hence, the observed effect of the down-regulation of T3SS genes might be an indirect effect of PtrB action [exsE may exert an effect on T3SS genes, as ExsE acts as an anti-anti-activator, which interacts with ExsC, a positive regulator of the type III secretion regulon [PA3035–PA3037 operon, where PA3035 encodes glutathione S-transferase, may be connected with transport functions, since such transferases are engaged in detoxification processes [Genes upregulated in response to PA2577 encompassed the B action . Similar regulon . Similarrocesses .PA2576 encoding a drug–metabolite transporter (DMT) from the EamA family PA2576. The action of PA2577 resembled other local regulators from the Lrp/AsnC family being divergently transcribed in respect to the target gene, e.g., AsnC from E. coli [P. putida [Agrobacterium tumefaciens [Rhodobacter capsulatus [Our in vitro and in vivo studies showed that PA2577 was directly involved in the regulation of the divergent gene  E. coli ,65, BkdR. putida , PutR inefaciens  or PutR E. coli. In the structure of the representatives of the EamA family, two identical parts comprising individual EamA domains could be distinguished. It is most probably an effect of the intra-protein domain duplication event during evolution [E. coli being an archetype for the family, but also PecM from Erwinia chrysanthemi [The DMT superfamily of proteins comprises 26 families . The Eamvolution . The funsanthemi ,69. Manysanthemi .P. aeruginosa ΔPA2576 mutant in the presence of polymyxin B and EDTA. This was connected with the increased sensitivity of the PA2576 mutant to agents acting on bacterial membranes. This linked the action of PA2576 with the proper maintenance of membrane functions, homeostasis and LPS biosynthesis through PA5006.Although we were not able to identify the cargo of the PA2576 transporter, our studies demonstrated its interactions with the putative lipoprotein PA3694 and putative kinase PA5006. These interactions may partially explain the observed disturbances in the growth of the P. aeruginosa was proposed  and further to the outer membrane [Based on the presented results, the model of the regulatory network engaging PA2577, PA2576 and the partners in proposed . PA2577 membrane . MsbA wamembrane . The Msbmembrane . A lipidmembrane . PA2576 membrane .ampDh3 and the PA3035–PA3037 genes  as the carbon source and leucine (10 mM) for leu− strains. For plasmid selection in E. coli, media were supplemented with 10 µg/mL chloramphenicol, 50 µg/mL kanamycin or benzyl penicillin at a final concentration of 150 µg/mL in liquid medium or 300 µg/mL in agar plates. For P. aeruginosa strains, the following antibiotics and concentrations were applied: carbenicillin (300 µg/mL), rifampicin (300 µg/mL), kanamycin  and chloramphenicol . For phenotype growth tests, M9 medium supplemented with EDTA in a concentration of 0.5 mM and polymyxin B in a concentration of 1 µg/mL was used.Bacterial strains used and constructed in this study are listed in E. coli cells were prepared with the use of the CaCl2 method and transformation was conducted according to a standard procedure [P. aeruginosa cells were prepared as described earlier [Competent rocedure . Compete earlier .All plasmids used and constructed in this study are described in P. aeruginosa PAO1161 WT or mutant strain with the pAMB9.37, pABB28.1 or pKGB8 derivatives.Overexpression of selected genes was examined by the transformation of P. aeruginosa PAO1161 ΔPA2577 and ΔPA2576 chromosomal mutants were obtained by the method of allele exchange [E. coli S17-1 cells with pMEB17 or pMEB164 plasmid (derivatives of suicide vector pAKE600). P. aeruginosa PAO1161 RifR was used as the recipient. After conjugation, selection and screening of transconjugants were performed by the growth on LB plates with rifampicin and carbenicillin. Removal of the vector was conducted by overnight growth in LB with 10% sucrose. Selection of colonies with exchanged allele was performed by PCR using primer pairs #3/#6 and #10/#13 (exchange . The don #10/#13 .600) in a spectrophotometer or with the use of Varioskan Lux Multimode Microplate Reader and SkanIt RE software  in the case of growth in 96-well plates.For tests of growth kinetics, liquid media were inoculated with strains propagated on plates. Overnight cultures were diluted 1:100 in a fresh medium and then incubated at 37 °C whilst shaken. When the experiment was performed in M9 medium, overnight cells grown in LB were washed twice with M9 medium and then used for inoculation. Bacterial growth was monitored by the measurement of optical density at 600 nm  system was used to analyze protein–protein interactions in vivo ,44. CyaAescribed . BACTH sMotility assays were performed as described previously . To stanhttp://www.ncbi.nlm.nih.gov/geo/ (accessed on 30 October 2021)) under accession number GSE186749.Strains obtained by transformation of PAO1161 with pMEB12 or pKGB8 were used for total RNA preparation. RNA isolation and sequencing as well as data analysis were performed essentially as previously described . Raw datnadB was used as the reference. RT-qPCR was also used for examination of the expression of PA2577 and PA2576 in the early (OD600~0.5) and late exponential (OD600~1.5) phases of growth of P. aeruginosa WT cultures and for PA2576 also in P. aeruginosa ΔPA2577 strain. The rpsL gene was used as the reference. Relative gene expression was calculated using the Pfaffl method [Expression changes for selected genes were confirmed by RT-qPCR using RNA isolated from independent cultures. In total, 1 µg of each RNA sample was used for reverse transcription  with the use of random hexamers. Three technical replicates of PA2577 overproducer and EV control were used. The l method . All oliP. aeruginosa ΔPA2577 strain carrying pMEB12 (araBADp-PA2577-flag) or pKGB8 vectors was grown in a medium containing 0.02% arabinose as the inducer. Additionally, it was used in the ChIP procedure with anti-flag antibodies . Data were processed essentially as previously [http://www.ncbi.nlm.nih.gov/geo/ (accessed on 30 October 2021)), under accession number GSE186746.ChIP was performed as previously ,82. P. a6-tagged PA2577 protein, E. coli BL21(DE3) strain was transformed with pMEB105. Cells were grown overnight in LB broth at 37 °C with 0.5 mM IPTG and then harvested by centrifugation. Phosphate buffer  supplemented with lysozyme at a final concentration of 1 mg/mL and 1 mM PMSF was used for resuspension of the pellet. The supernatant obtained after sonication and centrifugation was transferred onto Ni-agarose columns  and purification of the PA2577-His6 was carried out according to the manufacturer’s instructions. The gradient of imidazole  was used for elution. Purified proteins were dialyzed by overnight incubation in phosphate buffer containing 10% (v/v) glycerol and stored in small aliquots at −80 °C. Purification was monitored by SDS-PAGE with a Pharmacia PHAST gel system.To purify His6 was cross-linked with increasing concentrations of glutaraldehyde as previously described [w/v) SDS-PAGE gels. After overnight wet transfer onto nitrocellulose membranes , Western blot analysis was performed with anti-His6 mouse antibodies.Purified PA2577-Hisescribed . Samples6 was conducted in binding buffer (10 mM Tris-HCl (pH = 8.5), 10 mM MgCl2, 100 mM KCl, 0.1 mg/mL BSA). DNA was amplified with the use of #16/#21 primers for PA2576 promoter and #20/#21 primers for non-specific DNA. pMEB78 and empty pCM132 were used as templates. DNA was stained by ethidium bromide and visualized by UV light. PA2576p region encompassed the whole 132 bp intergenic fragment between PA2576 and PA2577. The complexes were separated on a 10% polyacrylamide gel in 0.5 × Tris-borate-EDTA (TBE) buffer.Electrophoretic mobility shift assay with purified PCR products and PA2577-HisE. coli DH5α carrying pPTOI derivatives with a promoter fused to the xylE reporter gene together with pMEB64 (QlacI-tacp-PA2577) or pAMB9.37 (QlacI-tacp) were assayed for catechol 2,3-oxygenase activity (the product of xylE gene). The experiment was performed as previously described [escribed  using ceescribed .cyaA fragment) was used [PA2577 and PA2576 genes were cloned in the pLKB2 and pKGB5 plasmids to obtain proteins fused with the T25 cyaA subunit as described in 2 method and used for transformation with pUT18C, containing random fragments of P. aeruginosa genomic DNA. The mixture obtained after the transformation procedure was washed with sterile water to discard the rich medium and, after that, plated onto M9 minimal medium supplemented with kanamycin, penicillin, 75 µM thiamine, 1% maltose, 40 µM X-gal and 0.1 mM IPTG. Plates were incubated for 5 days at 30 °C and all blue colonies indicating the interaction between T18 and T25 cyaA subunits and reconstitution of CyaA activity were re-streaked on the same medium. Plasmid DNA isolated from colonies that remained blue after re-streaking was used to perform the transformation of E. coli BTH101 cells carrying empty pLKB2 or pMEB122/pMEB67. The red color of colonies obtained on MacConkey plates for the transformation of cells carrying PA2576 or PA2577 and white colonies in the case of cells with an empty vector were presumed as the confirmation of identified interactant. Plasmid DNA containing a fragment of positively verified putative partners was sequenced. Genes encoding identified partners were then cloned into the BACTH vectors and interactions with full-length proteins were tested.For the search and screening of protein interactants, the available PAO1161 genome library constructed in the pUT18C vector   were used to test the growth capacity in different conditions of the analyzed PAO1161 WT, Δescribed ,87. P. a


Growing evidence suggests that ADHD, an early onset neurodevelopmental disorder, is associated with poor somatic health in adulthood. However, the mechanisms underlying these associations are poorly understood. Here, we tested whether ADHD polygenic risk scores (PRS) are associated with mid-to-late life somatic health in a general population sample. Furthermore, we explored whether potential associations were moderated and mediated by life-course risk factors. We derived ADHD-PRS in 10,645 Swedish twins born between 1911 and 1958. Sixteen cardiometabolic, autoimmune/inflammatory, and neurological health conditions were evaluated using self-report (age range at measure 42–88 years) and clinical diagnoses defined by International Classification of Diseases codes in national registers. We estimated associations of ADHD-PRS with somatic outcomes using generalized estimating equations, and tested moderation and mediation of these associations by four life-course risk factors . Results showed that higher ADHD-PRS were associated with increased risk of seven somatic outcomes  with odds ratios ranging 1.07 to 1.20. We observed significant mediation effects by education, BMI, tobacco use, and alcohol misuse, primarily for associations of ADHD-PRS with cardiometabolic outcomes. No moderation effects survived multiple testing correction. Our findings suggests that higher ADHD genetic liability confers a modest risk increase for several somatic health problems in mid-to-late life, particularly in the cardiometabolic domain. These associations were observable in the general population, even in the absence of medical treatment for ADHD, and appear to be in part mediated by life-course risk factors. Attention-deficit/hyperactivity disorder (ADHD) is an early onset neurodevelopmental disorder with an estimated prevalence of 5–7% in childhood . There iTwo recent reviews concluded that ADHD is robustly associated with obesity, sleep disorders, epilepsy, migraine, and asthma. Further, increasing evidence supports associations of ADHD with cardiovascular disease, neurodegenerative disorders, and certain autoimmune/inflammatory conditions , 7. SomeOne approach to partly circumvent these limitations is to study the effect of ADHD genetic liability in population-based samples that are untreated for ADHD, using polygenic risk scores (PRS), which capture the sum of an individual’s common autosomal genetic variant liability for ADHD based on independent genetic discovery data . Such apN = 52,080) were contacted for a telephone interview between 1998 and 2002 in the Screening Across the Lifespan Twin (SALT) study. In total, 44,919 individuals completed the SALT interview (response rate 85%). Study design and data collection has been described in detail elsewhere  = 1) across the p-value thresholds, retaining the first PC to derive a single (PC-)PRS used in subsequent analyses. This approach has been shown to maximize the amount of variance captured across PRS p-thresholds, whilst avoiding overfitting and maintaining a correct Type I error rate , mean centered and scaled), tobacco use (ever/never for snuff and cigarettes), and alcohol misuse . For alcohol misuse, we also included information regarding lifetime alcohol use disorder from the NPR . Life-course risk factors were assessed by self-report in the SALT interview and asked in a lifetime perspective.We identified four life-course risk factors previously associated with ADHD  as potenData collections in SALT/TwinGene and linkage to national registers were approved by the Swedish Ethical Review Authority in Stockholm. Informed consent was obtained from all participants.R2 was estimated for the full model  and the null model (not including ADHD-PRS). Percentage variance explained by ADHD-PRS for each outcome was calculated as the difference between the two (ΔR2). To evaluate differential associations by ascertainment source, we ran sensitivity analyses for self-reported and register-based outcomes separately. Further, we explored potential sex differences in the association of ADHD-PRS with each somatic outcome by running sex stratified analyses.We estimated associations between ADHD-PRS and each somatic health outcome separately using generalized estimating equations (GEE) implemented in the R-package DrGEE , and adjWe tested whether the four life-course risk factors  were associated with ADHD-PRS, using the same statistical model as above. As all factors showed evidence of association, we further tested for moderation and mediation effects by each life-course risk factors on the ADHD-PRS-outcome associations. Moderation effects were tested by including an interaction term to the main analyses  between ADHD-PRS and each life-style risk factors, in separate models. Finally, we ran a regression-based causal mediation analysis using the counterfactual approach  to explop-values using the Benjamini–Hochberg method to safeguard against multiple testing within each set of analyses. Data management was performed using SAS software version 9.4  and analyses ran using R 4.1.0 [All analyses were adjusted for birth year, sex, the first six PCs, and length of follow-up in register data . We obtained false-discovery rate (FDR) corrected  R 4.1.0 .N. The number of cases and prevalence rates for somatic health outcomes are presented in Table Among the 10,645 included individuals, 5587 (53%) were female. The mean age was 59 years (range 42–88) at self-report data collection, and 72 years (range 53–100) at the end of register-based follow-up (31st December 2013). In total, 1016 (10%) individuals had died and 9 (0.1%) emigrated after donating DNA in the TwinGene data collection (2004–2008) and before the end of register-based follow-up. Only 11 individuals had an ADHD diagnosis and/or an ADHD medication dispensation in register data, in line with evidence that ADHD is often underdiagnosed and untreated in older adult populations [p-values and ΔR2 are presented in Table R2 for the combined, register-based, and self-reported outcomes can be found in Fig. Associations between ADHD-PRS and somatic health outcomes defined by combined register and self-reported data are presented Fig. Descriptions of the four life-course risk factors and their associations with ADHD-PRS are presented in Table No interaction effects survived multiple testing correction. Results from the moderation analyses are presented in Table p < 0.05) after FDR correction. An overview of the mediation results and FDR corrected p-values can be found in Table Table We evaluated the role of ADHD genetic liability, as captured by PRS, on somatic health conditions in mid-to-late life in a general population sample. Our results suggest that polygenic risk for ADHD confers modestly increased risks for a range of somatic health problems in adulthood, particularly in the cardiometabolic domain. These associations were evident in a general population sample untreated and undiagnosed for ADHD. Further, we present novel, albeit tentative, findings to suggest that part of the association between ADHD genetic liability and primarily cardiometabolic outcomes may be mediated by life-course risk factors .Higher ADHD-PRS were associated with four out of seven cardiometabolic outcomes, including obesity, peripheral vascular disease, heart failure and cerebrovascular disease. There is an extensive body of literature linking ADHD to obesity . Furtherrg 0.16 and 0.23, respectively) were reported in one prior study, whereas the association to Type 1 diabetes was not found to be statistically significant [Overall, we found less evidence for genetic links between ADHD-PRS and diseases in the autoimmune/inflammatory and neurological domains. Among the autoimmune/inflammatory diseases, ADHD-PRS were associated with a small increased risk for type 1 diabetes and rheumatoid arthritis, but not psoriasis or inflammatory bowel disease. Some prior studies have found associations of clinical ADHD with type 1 diabetes and rheumatoid arthritis, both within-individual and across relatives, suggesting a possible common genetic vulnerability between ADHD and certain autoimmune diseases , 44. Hownificant . More rerg ~ 0.2 to 0.4) [Within the neurological domain, migraine was the only outcome associated with ADHD-PRS, with no significant associations observed for epilepsy, dementia, Parkinson disease, or sleep disorders. Although there are well-established phenotypic links between ADHD and certain neurological diseases , evidence of genetic overlap with ADHD are mixed . Large-s to 0.4) , 46. A q to 0.4) . Togethe to 0.4) , in part to 0.4) .We found no evidence for moderation of ADHD-PRS associations by the investigated life-course risk factors , with minor differences observed between exposure groups. The few available studies investigating interaction effects using ADHD-PRS have focused on ADHD as the outcome and largely reported null findings , much liIn contrast, we did find support for a mediating role of lower education, higher BMI, tobacco use, and alcohol misuse, on the relationship between ADHD-PRS and several somatic outcomes. Mediation effects were observed for nearly all cardiometabolic outcomes, for multiple mediators. For example, the main effect of ADHD-PRS on peripheral- and cardiovascular diseases showed significant mediation by all four life-course risk-factors. Further, BMI and alcohol misuse showed evidence of mediation on all significant ADHD-PRS main effects in the cardiometabolic domain . Further, BMI also mediated part of the association of higher ADHD-PRS with type 1 diabetes and migraine. The mediating effect of higher BMI across disease domain could either reflect the broad systemic effect that obesity can have on health and the strong link between ADHD and obesity. It may also in part be because BMI was the only mediator modeled as a continuous variable, thus increasing the power to detect even very small mediation effects. It is important to note that the life-course risk factors were asked about in a lifetime manner, meaning they did not necessarily precede the outcomes and mediation effects should therefore be cautiously interpreted. Further, it is important to note that both the main and mediation effects of ADHD-PRS were modest across all three disease domains, and our findings are thus far from clinical utility . This is unsurprising given that the current ADHD-PRS only explains about 5% of variance in ADHD diagnosis, and considerably less in related traits and comorbidities , 20. HowOur results add to a growing body of evidence suggesting that ADHD common variant genetic liability confers a small increased risk for several mid-to-late life somatic health condition , 22. MorThere are several strengths but also important limitations of this study. First, assessment of somatic health outcomes by both self-report and national medical registers represents an advantage of this study, yet the two data sources may capture partly different case definitions. Self-report provides information on somatic health problems that are untreated or mostly treated in primary care (which we did not have information from), and for symptoms below the diagnostic threshold. Conversely, register data from specialist care captures more severely affected patients and has excellent coverage for diseases requiring specialist treatment  . As suchOur study suggests an overall pattern whereby higher ADHD genetic liability is linked to a modest risk increase for several somatic health problems in adult life, particularly in the cardiometabolic domain. These associations were observable in the general population, even in the absence of medical treatment for ADHD, and may in part be mediated via life-course risk factors.Supplementary materials


An important aspect of hydrocarbon drilling is the usage of drilling fluids, which remove drill cuttings and stabilize the wellbore to provide better filtration. To stabilize these properties, several additives are used in drilling fluids that provide satisfactory rheological and filtration properties. However, commonly used additives are environmentally hazardous; when drilling fluids are disposed after drilling operations, they are discarded with the drill cuttings and additives into water sources and causes unwanted pollution. Therefore, these additives should be substituted with additives that are environmental friendly and provide superior performance. In this regard, biodegradable additives are required for future research. This review investigates the role of various bio-wastes as potential additives to be used in water-based drilling fluids. Furthermore, utilization of these waste-derived nanomaterials is summarized for rheology and lubricity tests. Finally, sufficient rheological and filtration examinations were carried out on water-based drilling fluids to evaluate the effect of wastes as additives on the performance of drilling fluids. Drilling operations are conducted to obtain oil and gas from natural reservoirs deep underground . To faciDrilling fluids must address several challenges encountered during the drilling process. For instance, the drilling structures erected during the process are made of metal, and are thus susceptible to corrosion, which in turn affects the overall drilling operation . A drillOther possible scenarios in drilling operations include equipment failure during wellbore completion. Wellbore completion is the process of preparing a wellbore before the production stage, to ensure that the desired hydrocarbons flow out of the formation into the wellbore, and then out . In thisSharma et al. observed that tamarind gum and polyanionic cellulose showed better rheological properties and filtrate loss control performance in an oil well, and also significantly reduced formation damage . Meng etFurthermore, Ismail et al. established that henna leaf extract and hibiscus leaf extract enhanced rheology and filtration properties of water-based drilling fluid in comparison to a common additive used in the industry . Oseh etThe drilling industry currently uses many additives that provide satisfactory drilling fluid performance. However, these materials have been found to be hazardous, either to the workforce operating on the site or to the environment. Substantial research has been conducted on possible alternative drilling fluid additives that fulfill two conditions: first, that the additive provides the properties required of drilling fluids; and second, that it is environmentally friendly, biodegradable, and sustainable. This paper reviews the research undertaken using various environmentally friendly waste-generated additives in water-based drilling fluids. In particular, the role of these additives on rheological properties, such as plastic viscosity, yield point, gel strength, filtrate loss, and mud cake thickness, is evaluated. Additionally, as a noteworthy aspect of drilling fluids, the impact of various nanomaterials as additives in lubricity tests us summarized.The principal functions of drilling fluids include removal of drill cuttings and cleaning of the wellbore, lubricating and cooling the drill bit and string, maintaining wellbore formation, and preventing well blowout ,20. ThusThe mud density or weight is an important property of drilling fluids that functions to improve wellbore stability and maintain formation pressure. According to Das and Chatterjee , low mudViscosity measures the internal resistance of drilling fluids, whereas PV is the flow resistivity caused by friction between the solid particulates in drilling fluids and fluid layers . PV is dYP is defined as the measured degree of shear thinning performance of non-Newtonian drilling fluids. It is the ability to carry drill cuttings in suspension while circulating in the wellbore and out of the annulus. Hence, drilling problems such as differential sticking can be prevented . AccordiGel strength (GS) measures the forces of attraction between particles in static conditions, unlike YP which measures them in dynamic conditions . TherefoFiltration or fluid loss measures the amount of liquid that permeates a solid mud cake formation. According to previous researchers , drillinThe global population, which is currently 7.8 billion and growing at 1.1% per year, relies on the consumption of the Earth’s natural resources . Waste mWaste material includes municipal solid waste (MSW), which comprises common items consumed and discarded by the public, and represents the fastest growing form of waste due to its prevalence in urban society . In a reOther types of waste are produced by a range of sources, including domestic and commercial; ash; animals; biomedical and construction industries; and sewers. These wastes may comprise industrial solid waste, biodegradable and non-biodegradable waste, and hazardous waste .Some of these types of waste pose a serious threat to the environment and human health. Clinical waste, which is produced by medical clinics, hospitals, and laboratories, carries the risk of infection and may spread disease if not appropriately managed ,41.Electrical and electronic waste (E-waste), from electronic equipment such as cables, wires, cords, and batteries, releases dangerous substances, and thus causes serious harm to those who contact it, particularly workers in the recycling industry ,43. AddiFood waste is a major global issue that is caused by factors including poor food processing operations and management, inadequate household planning of food consumption, and over preparation of food in the food and beverage industry ,45. NeveA significant amount of research has been conducted regarding the use of food waste in the oil and gas drilling industry . For insFurthermore, Al-Hameedi et al. recognized that grass, hay, and palm leaves are also viable candidates. The study verified that food waste can be repurposed to promote an environmentally friendly operation of the oil and gas drilling industry . Figure The oil and gas drilling industry uses additives in drilling fluids for a variety of uses, such as pH control, and to provide rheological properties, such as plastic viscosity, gel strength, and yield point ,53. ThesRecent studies have shown the impact of various waste-derived additives for efficient rheological properties in drilling fluids . For exaMoreover, Murtaza et al. demonstrated the use of environmentally-friendly okra as a viable alternative additive in drilling fluids. The performance of okra as an additive was evaluated with the absence and presence of clay in drilling fluids. Comparatively, the incorporation of okra in clay-based drilling fluids presented a greater improvement in the rheological properties compared to that in clay-free drilling fluids. In clay-based drilling fluids, the addition of 2 and 3 g of okra resulted in an increase in plastic viscosity (PV) of more than 100%, compared to the addition of 2 g of starch, which only yielded a 45.7% increment. Increasing the concentration of okra also led to an increase in the yield point of drilling fluids. However, was observed that starch is more efficient in improving the yield point than okra. Fluid loss was evaluated at different concentrations and observed to be reduced at different proportions for each concentration . In addiSimilarly, Ghaderi et al. proposed sustainable saffron purple petals (SPP) as an eco-friendly alternative for additives in drilling fluids. The addition of SPP powder in drilling mud resulted in an effective increase in PV values. As the concentration of SPP powder increases, the PV value also increases. Additionally, the introduction of SPP powder to drilling mud also dramatically enhances the yield point compared to that of base mud. The incorporation of SPP powder into drilling mud demonstrated excellent filtrate loss, whereby the filtrate volume was reduced gradually with increasing concentration of SPP powder. The addition of SPP powder in the drilling mud also resulted in the reduction of mud cake thickness compared to that in base mud ,60.The above-mentioned studies have demonstrated the effectiveness of food waste in drilling fluid additives as a substitute for the environmentally hazardous materials currently in use within the industry . In thisBentonite is a clay material that is naturally composed of sodium montmorillonite and minor quantities of minerals. It is used in a variety of applications due to its absorption and adsorption capabilities . For insIn the presence of water, bentonite hydrates and swells to form a thixotropic gel, i.e., a mud cake, thus protecting the well formation from invasion, which causes the loss of fluids to permeable formations. However, mud cakes formed by bentonite have been found to be highly detrimental to the productivity of drilled wells. This is due to the inefficient removal of protective mud cakes that is undertaken to restore performance of wells . AccordiMost importantly, because bentonite clay contains montmorillonite, a crystalline structure that forms the clay, bentonite precipitates when water is added . This isMagzoub et al. have studied bentonite compounds comprising different primary elements, such as calcium (Ca), potassium (K), and sodium (Na), and found that sodium bentonite is commonly utilized in drilling fluids. In contrast, calcium is rarely used due to its unsatisfactory rheological properties. However, this study used sodium to activate the calcium bentonite to improve its performance .Karagüzel et al. have found that sodium and calcium bentonites in combination with soda and MgO additives show enhanced swelling properties, lower filtrate loss, and increased viscosity at favorable concentrations. An important fact to note is that although sodium and calcium bentonites were used as mud viscosifiers and fluid loss reducers, they did not qualify as good drilling fluids. This finding highlights the importance of choosing suitable additives that can effectively enhance the properties of a drilling fluid .The disadvantages of bentonite have been studied, leading to the formulation of nanomaterial-based drilling fluids, and resulting in significant improvements in wellbore cleaning properties while successfully maintaining optimum viscosity and density . CompareNanomaterials are manufactured substances that have a size ranging from 1 to 100 nanometres (nm), and therefore are utilized in extremely small dimensions ,91. NanoTo facilitate the drilling of a borehole, drilling fluids imparts a vital factor for a successful drilling operation; that is, the fluids help remove the drill cuttings and fragments from the drilling area and the wellbore.Nanomaterials have an extremely high surface area to volume ratio due to their nano-sized particles. Therefore, WBDF containing nanomaterial active agents possesses improved physical and chemical sensitivity, which enhances its performance efficiency compared to that of OBDF . In addiFurthermore, nanomaterials have also been used as lubricants to reduce friction between the wellbore and drill string, which consequently reduces the likelihood of a stuck pipe. 2), the rheological, hydraulic, and filtration properties of WBDF are effectively improved [The pioneering works on the significant usage of nanomaterials in drilling fluids were undertaken by Abdo et al. . In enhaimproved . In a reimproved . The addimproved . Gautam improved . In summimproved .For example, Kasiralvalad et al. found that adding traces of nanomaterials to the drilling fluid, thereby making it a nanofluid, played an essential role in enhancing the mud cake quality, and reduced the sticking of the pipe to the reservoir, promoted good borehole stability and reservoir protection, and increased recovery of both oil and gas products. This was possible due to the modifications to the fluid caused by the nanoparticles, which aided in its superior performance .Another reflection of this finding was provided by the study of Li et al., in which the authors established that nanomaterials helped to improve the mud cake quality and reduced loss circulation. In addition to these qualities, they also found that when nanomaterials were used as viscosifiers, emulsifiers, and lubricants, they improved the qualities of borehole cleaning, borehole stability, and reservoir protection, and enhanced oil and gas recovery .Salih et al. emphasized the concentration of nanomaterials, and found that the rheological and filtration properties of the drilling fluid were superior at a low concentration compared to the inferior performance achieved at a higher concentration. Other researchers analyzed noteworthy parameters related to nanomaterial usage . This re3O4-carboxymethyl cellulose (CMC) nanocomposite as a fluid loss control additive in drilling fluids. In this study, the effect on rheological properties of drilling mud with and without salt was studied. The study observed that the utilization of the nanocomposite enhances the fluid viscosity of the drilling mud under both conditions. Based on the study results, in general, the yield point of the drilling fluid is further increased with increasing concentration of the nanocomposite. It was observed that the integration of the nanocomposite into the drilling fluid allows for the production of a thinner filter cake in comparison to the CMC mud system [In addition to waste-derived materials, recent studies have presented nanomaterials as promising alternative for use as additives in drilling fluids. As an example, Kamali et al. assessed the effects of Fed system . In addiSaboori et al. highlighted the importance of adding the appropriate concentration of additives to the drilling fluid for improved properties. They investigated the addition of varying concentrations of copper oxide (CuO), also called polyacrylamide nanocomposite, to a water-based drilling fluid. They found that increased concentration significantly minimized fluid loss and filter cake thickness in comparison to the absence of nanocomposites. They also observed higher viscosity, higher thermal conductivity, and a favorable filter cake porosity .Another important distinction was made by comparing drilling fluid performance between salty and non-salty water. It was found that both types displayed the best performance under certain conditions, and that a specific salt concentration may result in the best performance for both salty and non-salty water. This study again showed that additives play a vital role in the performance of a drilling fluid . Figure A compelling recent study carried out by Lekomtsev et al. utilized tools including an Extreme Learning Machine (ELM) and Particle Swarm Optimization-Least Square Support Vector (PSO-LSSVM) to investigate the effect of various nanoparticles on the filtration of a volume of drilling fluids. This research showed a decrease in the amount of filtration volume with the increase in the weight percentage of the nanoparticles. Among the parameters evaluated, the study indicated that nanoparticle concentration had the greatest impact on the filtration volume and mud cake thickness of drilling fluids ,111.Al-Zubaidi et al. studied nano-Iraqi clay and other nanomaterials, such as graphene and magnesium oxide (MgO), in various concentrations and separately combined with commercial nano-bentonite to observe the performance of the fluid. The addition of MgO with nanomaterials resulted in an improved filter loss and yield point. This demonstrates that a wide range of additives with appropriate concentrations can improve the performance of drilling fluids .Graphenaceous materials have been extensively used in drilling fluids because they promote and enhance the rheological properties of these fluids. Kosynkin et al. found that the use of a graphene oxide additive in water-based drilling fluids improved its filtrate loss properties, significantly lowering the filter cake thickness and fluid loss, and screening enhanced shear thinning and thermal stability. It was also observed that graphene in both flake and powdered form was a contributing factor to the improved performance. This study indicates that the presence of an additive influences the properties of a drilling fluid, and shows how the form of the additive can contribute to value-added properties .2/polyacrylamide nanocomposite as an additive in water-based drilling fluids, and found a significant reduction in fluid loss and mud cake thickness with its use, as observed in Sadeghalvaad and Sabbaghi studied a TiOPatel et al. observed that conventional water drilling muds exposed to water sensitive shale cause the shale to absorb water from the mud, resulting in problems during operation. Therefore, the water-based muds must contain additives to effectively inhibit the shale. Salt compounds were used to inhibit the shale. However, the high concentrations of the salt compounds affected the surrounding ecosystems. The concentration was altered, and the shale was successfully inhibited . This stSimilarly, Qu et al. investigated polyoxyalkyleneamine (POAM) as a potential additive and found that POAM improved the shale inhibition capabilities in water-based drilling fluids . As an a2 nanoparticles added to water-based drilling fluids enhance inhibition, and filtrate loss and rheological properties. The added benefit is that the preparation of SiO2 nanoparticles has a low cost due to their common methods of preparation [In the drilling industry, many researchers have studied nanomaterials and found a wide range of chemicals that can improve the properties of drilling fluids. The clear advantage of using nanomaterials is that the amount required is very small . Hence, paration .2 nanoparticles is below 1% w/v in shale inhibition, thus constituting a very small, and hence economical, concentration [Similarly, Taraghikhah et al. found that the optimal concentration of SiOntration .2 nanoparticles as an additive in water-based drilling fluids. They found that SiO2 nanoparticles increased the viscosity of the fluid, thereby allowing it to more efficiently carry drill cuttings from the wellbore. This ensures the wellbore is clean and, therefore, does not pose challenges when the drill needs to be removed or maintenance work is required [Gbadamosi et al. investigated SiOrequired .2 nanoparticles with xanthan gum as a base in water-based drilling fluids, and found an increased yield point, superior hole cleaning ability, reduced filtrate loss, and more efficient lubrication of the drill bit during operation compared to drilling fluids without SiO2 [Al-Yasiri et al. also investigated the use of SiOout SiO2 .2O3), titanium dioxide (TiO2), SiO2, and CuO in bentonite, and their effects on water based drilling fluids. They found that the combined additives improved overall rheological properties and gel strength at low concentrations in comparison to the base fluid without nanoparticles [Bayat et al. studied four nanoparticles types, namely, aluminum oxide  nanoparticles on the rheological properties of non-damaging drilling fluid (NDDF). In comparison to base NDDF, NDDF incorporated with ZnO nanoparticles exhibited higher shear stress and viscosity. The addition of ZnO nanoparticles to NDDF helped overcome the issue of NDDF degradation through stabilization of viscosity at higher temperatures. Temperature sweep test measurements indicated a good operational temperature range of base NDDF was between 70 and 80 °C. NDDF containing ZnO nanoparticles exhibited improvement in fluid loss control. However, it was observed that an increase in pressure resulted in a decrease in fluid loss . Table 2One of the significant characteristics of drilling fluids is lubricity. Lubricity is required in order to reduce the friction due to the continuous contact between the wellbore and drilling string in the horizontal and directional wells . There aFarahbod et al. presented a study of the thermo-physical properties of a drilling fluid incorporated with nanoparticles to examine the capability of drilling fluids to transfer heat. The use of nanoparticles, including titanium dioxide nanoparticles and CNT, were suggested because nanoparticles possess a high specific surface area, which may increase the rate of heat transfer . The stuIn drilling fluids, lubricity reduces the torque and drag force . TypicalHusin et al. reported a torque reduction of 20% with the use of a lubricant concentration of only 1% . Some ofStudies have reported various types of additives used as a lubricant for drilling fluids, including modified vegetable oils and refined polyols . The comIn recent decades, there has been growing interest in the use of nanomaterials as lubricant additives. This interest has been motivated by the movement in the industry toward the use of water-based drilling fluid due to the environmental concerns associated with the use of oil-based and synthetic drilling fluids . The novBased on the results of recent studies, the usage of environmentally friendly additives such as bio-wastes has significantly improved the performance and functionality of drilling fluids compared to commercial GO.However, several challenges must be addressed before these bio-wastes can be applied and commercialized at a larger scale in the oil and gas industry. One of the key issues is that raw waste materials and waste-derived nanomaterials may contain high impurities, thus necessitating an additional purification process. Therefore, future studies and exploration must be carried out to improve the yield and characterization of waste-derived nanomaterial production. A schematic illustration of the challenges related to the collection of waste materials and their recycling, and the interconnected role of consumers in society, is presented in In addition, it is also important to examine novel waste derivatives by conducting aging tests and experimental studies under HPHT conditions to study the degradation of the environmentally friendly additive. In addition, the environmental impacts of waste-derived nanomaterials should be highlighted. These waste additives can be added to and optimized in OBDF and SBDF formulations, in addition to those of WBDF. Furthermore, a thorough comprehensive quantitative analysis of different types of green additives and their performance can be conducted to determine the best rheological improvements. Hereafter, the role of environmentally friendly waste-derived additives will have a major role in the preparation of novel green additives for drilling fluids. It is recommended that future research focuses on identifying the green additive that optimally improves the significant rheological and filtration properties of drilling fluids. Therefore, a breakthrough can be achieved by improving the efficiency of drilling operations while reducing any harmful risks to the environment and the health of personnel.To implement the use of waste-derived materials, more research is needed to achieve a better understanding for application in the oil and gas industry. Waste materials such as food waste have the potential to be utilized as an alternative to harmful and toxic additives that are conventionally used in drilling operations. To date, a variety of waste-derived materials have been explored due to their potential use as additives. These materials include food waste, such as durian rind, and plant-derived wastes, such as black sunflower seeds. The role of waste-derived nanomaterials, and the key features with the potential to improve the efficiency of drilling performance, are presented in A comprehensive investigation of the interactions between waste-derived materials and content of drilling fluids, such as bentonite, should be undertaken.Cost-effectiveness of waste material usage requires more attention prior to commercialization to ensure consistency in generating drilling fluids with improved rheological properties.In-depth analysis is required to develop extensive methodologies for the production of additives based on waste-derived materials.Future studies should consider the analysis of the lubricity of drilling fluids using waste-derived materials. Extensive analysis should be undertaken to examine the morphological properties of drilling fluids.The potential to convert waste materials into nanomaterials, and the reproducibility of the conversion, should be considered for a variety of applications.A comprehensive quantitative analysis of nanomaterials used in drilling operations is necessary. Particular focus is required to determine optimum concentrations to improve conservation of resources.More studies should strive to investigate the mechanisms of interaction between nanomaterials and other additives present in drilling fluids.A comparison of drilling fluid optimization between water-based drilling fluids using nanomaterials, and synthetic and oil-based drilling fluids, should be undertaken. The comparison should be conducted in relation to conventional base fluids subjected to high temperature and pressure conditions.Recommendations of this study are as follows:The increased production of waste materials is a significant concern due to their effect on public health and the environment. Mismanagement of food waste, in particular, has become a major global issue, thus prompting the need for better solutions that use these materials in different applications. Among various applications, food waste can be considered to be a sustainable alternative for additives in drilling fluids used in the oil and gas drilling industry. Chemical additives to drilling fluids are necessary components to facilitate drilling operations by enhancing the fluids’ properties, including rheology and filtrate loss. Studies have demonstrated that waste-derived materials, including food waste, have the potential to provide an environmentally safe alternative to toxic conventional chemical additives used in water-based drilling fluids. The materials summarized in this review include food waste and waste generated from plants. The efficiency of these materials was evaluated in terms of their effects on the yield point, plastic viscosity, filtrate loss, and mud cake thickness.Nanomaterials are viable alternative additives for drilling fluid application. Nanomaterials can be used economically due to the small concentrations required for their efficient use in drilling fluids. Based on the summarized studies, quantities less than 1 g are sufficient to generate changes in the lubricity of drilling fluids. The lubricity of drilling fluids is a property that is considered to be necessary to ensure smooth drilling operations. For water-based drilling fluids, in particular, lubricant additives are required to provide better lubrication and thus reduce friction in drilling operations.


The new index’ ability to detect instructed simulators (n = 242) and non-credible adults with ADHD (n = 22) was subsequently examined using ROC analyses. Applying a conservative cut-off score, the new index identified 30% of participants instructed to simulate ADHD while retaining a specificity of 98%. Items assessing supposed symptoms of ADHD proved most useful in distinguishing genuine patients with ADHD from simulators, whereas inquiries into unusual symptom combinations produced a small effect. The CAARS Infrequency Index (CII) outperformed the new infrequency index in terms of sensitivity (46%), but not overall classification accuracy as determined in ROC analyses. Neither the new infrequency index nor the CII detected non-credible adults diagnosed with ADHD with adequate accuracy. In contrast, both infrequency indices showed high classification accuracy when used to detect symptom over-report. Findings support the new indices’ utility as an adjunct measure in uncovering feigned ADHD, while underscoring the need to differentiate general over-reporting from specific forms of feigning.As attention-deficit/hyperactivity disorder (ADHD) is a feasible target for individuals aiming to procure stimulant medication or accommodations, there is a high clinical need for accurate assessment of adult ADHD. Proven falsifiability of commonly used diagnostic instruments is therefore of concern. The present study aimed to develop a new, ADHD-specific infrequency index to aid the detection of non-credible self-report. Disorder-specific adaptations of four detection strategies were embedded into the Conners’ Adult ADHD Rating Scales (CAARS) and tested for infrequency among credible neurotypical controls (The online version contains supplementary material available at 10.1007/s00702-021-02318-y. Growing recognition of adult manifestations of attention-deficit/hyperactivity disorder (ADHD)   and examinees feigning general psychological disorders (d = 2.65). Sensitivity of the Ds-ADHD in detecting feigned ADHD was 75%, specificity was 97%.Similar to the ASIS’ infrequency scale, Robinson and Rogers  based thRobinson and Rogers  concludeThe present study aimed to develop new ADHD-specific infrequency items by adapting detection strategies formulated by Rogers , includiNeurotypical Control Group The Neurotypical Control Group was recruited from a pool of panel members registered with a Dutch online platform. This website invites interested members of the public to take part in online studies in exchange for financial reward. Invitations to partake in the present study were accepted by 1577 adults from the Netherlands. Reconcilable with an average drop-out rate of 30% reported for online studies . Galešić , 460 adu Galešić , these pT-Scores on at least one DSM Scale of the CAARS. T-Scores equal to or above 80 are expected to occur very infrequently among honest-responding, healthy adults who exert adequate effort during testing and are thus considered indicative of non-credible responding by the instruments’ authors  presented with significantly elevated range of 40 years and a median absolute deviation (MAD) of 10 years . The number of male  and female  participants was balanced. Three volunteers (0.30%) did not disclose their gender. Participants in this group reported an average of 13 years spent in education (MAD = 3). Table The median age of the 1001 remaining credible controls equaled 49 years, with a Overreporting Controls were significantly younger  than Credible Controls  and more commonly male (72.20%) than female (27.80%). Gender distribution therefore differed between credible and over-reporting controls, though the difference did not reach statistical significance . The groups were comparable with regard to their years spent in formal education  or the GET  and were thus removed from the pool of credible patients. Like Overreporting Controls, these Non-Credible Patients were excluded from the development of the new index. Four adults with ADHD (3.01%) were excluded as they completed neither the TOMM nor the GET. Seven additional patients with ADHD were excluded due to missing  or incomplete  data on the CAARS. In total, 100 participants remained in the Credible ADHD Group and 22 individuals formed the Non-Credible ADHD Group.Credible ADHD Group differed significantly from Credible Controls in age , but not gender distribution (χ2 (2) = 0.747, p = 0.688) or education . Compared to Overreporting Controls, the Credible ADHD included a greater percentage of female participants . Age  and education  did not differ between the groups.The z = 0.294, adjusted p = 1.00), gender distribution , and education . Both credible and non-credible patients with ADHD most commonly met diagnostic criteria for the combined subtype , with the inattentive subtype being less common . Two credible patients (2%) had been given a diagnosis of the hyperactive subtype, whereas no subtype was specified for nine cases (9%). Sixty-two percent of participants in the Credible ADHD Group reported psychiatric or neurological comorbidities, most commonly mood (n = 46) or anxiety (n = 16) disorders . Occurrence of more than one comorbid disorder was common, with 21 participants having received two additional diagnoses alongside ADHD and five adults having been diagnosed with three or four comorbidities. A similar picture emerged among non-credible adults with ADHD, where 68% of participants reported at least one relevant comorbidity. Given the high prevalence of comorbidities among adults with ADHD  or incomplete  data on the CAARS. Fifteen simulators (5.28%) were excluded as they reported psychiatric disorders other than ADHD in the course of testing. Reported symptoms of ADHD , on the other hand, were not considered a criterion justifying exclusion from the study.p < 0.01 in all cases). As the majority of simulating participants was female , the gender distribution in this group also differed from the Credible (χ2 (1) = 42.625, p < 0.01) and Overreporting Control Groups (χ2 (1) = 16.842, p < 0.01) as well as the ADHD Groups (χ2 (1) = 12.40, p < 0.01 for the comparison with credible patients; χ2 (1) = 10.402, p = 0.01 when comparing simulators to non-credible patients). In terms of education, simulators differed from credible participants in the control group  and the patient group , but not from over-reporting controls  or non-credible patients with ADHD .As shown in Table ADHD Symptom Severity Severity of both past and present ADHD symptomatology was assessed by means of self-report. Childhood symptoms of ADHD were measured using the Wender Utah Rating Scale (WURS-K)   In their long form, the Conners’ Adult ADHD Rating Scales (CAARS)  As part of this study, 15 new items were introduced to the long form of the CAARS. We denote this expanded version CAARS-ACI to allude to the new infrequency index: the ADHD Credibility Index (ACI).The newly written items represented disorder-specific adaptions of detection strategies used in existing tests of malingering and deception   at the University of Groningen. All participants in the Control Group gave written informed consent and were subsequently asked for anamnestic information including age, sex, and educational attainment. Additionally, participants were asked about any history of psychiatric or neurological disease, as well as pharmacological treatments affecting the central nervous system. They were then instructed to complete all self-report measures  honestly and to the best of their ability.ADHD Groups Having given informed consent, adults with ADHD were tested individually in a quiet room on clinic premises. They were assured that all data collected as part of the study would be analyzed anonymously and that the results would not affect their clinical assessment or treatment. No reward was offered for participation in the research project. Patients underwent a comprehensive clinical assessment, which encompassed self-report questionnaires, standardized measures of cognition, as well as the previously described validity tests. Testing took approximately 2 h, divided into two parts to avoid potential effects of fatigue . Volunteers were explicitly asked to feign ADHD in a realistic manner by providing believable answers . This was further incentivized by introducing the chance of winning a tablet PC if they were the one participant who feigned the condition most convincingly. In actuality, the PC was awarded to a randomly chosen participant; that is, irrespective of test performance. Following the assessment, which took approximately 70 min, participants were debriefed and instructed to stop feigning the disorder. Additionally, they were asked whether they had followed the given instructions. All participants answered in the affirmative.Item Selection, Calculation of ADHD Credibility Index (ACI) Scores, and Determination of Cut-Off Score In line with the approach first described by Suhr et al.  fell. Herein, we considered effects of both age and sex by conducting non-parametric significance tests of group differences and providing separate summary statistics on ACI scores.To allow for a dichotomous distinction between endorsed and non-endorsed items, responses given on the CAARS’ four-point scale were rescored, such that items endorsed with “0” or “1” were coded 0 (i.e. not endorsed). Responses of “2” or “3” were recoded as 1 . Each participant’s score on the ADHD Credibility Index was calculated by summing up the scores on the new items which had been endorsed infrequently by patients with ADHD and control participants alike. Again, in accordance with the approach taken by Suhr et al. , the CAAAssociation with Symptoms of ADHD To examine whether symptoms of ADHD were associated with elevated scores on the ADHD Credibility Index, we considered T-Scores above 65 on the DSM Scales indicative of clinically relevant ADHD symptomatology. This diverges from the CAARS manual, which suggests T-Scores above 70 or 75 to signal relevant symptomatology, but allows for comparability with the CII .Utility of the ADHD Credibility Index in the detection of non-credible symptom report The ADHD Credibility Index’ utility in discriminating genuine cases of adult ADHD from non-credible responding was examined in a series of ROC analyses.Simulation design and non-credible patient data In a first step, we determined the ADHD Credibility Index’ ability to discern the ADHD Group from the Simulation Group. Second, the ACI was used as a criterion distinguishing credible from non-credible adults with ADHD . The same analyses were run using the CAARS’ DSM Scales and the CII as criteria, such that the ACI’s performance could be compared to that of suspect T-Score elevations and the CII.Concordance with existing validity indicators We conducted a ROC analysis to investigate whether the ADHD Credibility Index was useful in detecting over-report on the DSM Scales  in the complete sample collapsed across groups. Agreement between the ACI and existing CAARS validity indicators was also determined. Specifically, the ADHD Credibility Index was compared to T-score elevations equal to or above 80 on the DSM Scales, to scores equal to or above 8 on the Inconsistency Index, and to suspect results  on Suhr’s Infrequency Index (CII).As depicted in Fig. N = 1383), internal reliability of the new twelve-item index was high (Cronbach’s α = 0.94).With twelve items having been selected, each of which was to be rated on a four-point scale, possible scores on the ADHD Credibility Index ranged from 0 to 36. Summary statistics for the ACI are presented in Table n = 997) and 94.7% of credible adults with ADHD (n = 90) produced a score at or below 21 on this index. No gender-specific differences in ACI were noted . However, results of a Kruskal–Wallis test showed statistically significant differences in ACI scores between age brackets . With the exception of 18- thru 29-year-olds, whose ACI scores did not differ significantly from 30- to 39-year-olds , post hoc tests revealed significant differences between all age groups .As illustrated in Appendix 2 in ESM, 99.7% of credible controls ]. Considering each subset of ACI items individually, the largest effect could be noted for inquiries into supposed symptoms. Items assessing exaggerated symptoms or selectivity of symptom reports yielded comparable results. The smallest effect emerged for the subscale tapping unusual symptom combinations. Effect sizes are summarized and illustrated in Appendix 3 in ESM.p < 0.01, 95% CI ]. The ACI thereby outperformed the DSM Inattention (E) Scale and the DSM (G) Total in the detection of simulators, whereas the DSM Hyperactivity/Impulsivity (F) Scale yielded results comparable to those of the ACI ].Applying a cut-off score of 21, the ACI correctly identified 71 simulators (30.34%) at a specificity of 98.50% ] or its individual subscales were considered (see Appendix 3 in ESM). Indeed, no participant in this small subset of non-credible patients scored above the cut-off value on the ACI. Six non-credible adults with ADHD (27.27%) produced suspect scores on the CII. Specificity of the CII was 69.00% . To this end, we collapsed all groups into one and split the combined sample into credible and over-reporting participants. In a first analysis, participants were considered over-reporters if their T-Score on any DSM Scale exceeded 80. Using the ACI as the metric predicting over-report, ROC analysis showed an AUC of 0.941 . Repeating the same analysis for over-report on individual DSM scales showed comparable results for the detection of over-report on the DSM Hyperactivity/Impulsivity (F) Scale and the DSM Total (G). The smallest AUC could be noted for the DSM Inattention (E) Scale (see Table 4). Suhr’s Infrequency Index (CII) outperformed the ACI in the classification of over-report. Using the CII to detect over-report on any given DSM scale resulted in an AUC of 0.966 . Sensitivity and specificity of the ACI and CII in detecting over-report on any DSM scale are presented in Table r et al. , we inveAgreement with existing CAARS validity indicators To examine the agreement of established validity indicators and the new infrequency index, we contrasted classifications based on T-Scores exceeding 80   and over-reporting controls (55.56%) . Once evaluated for infrequency among credible adults with ADHD and their neurotypical counterparts, twelve of fifteen newly written items remained and were summed to form the ACI. Four subscales, all corresponding to the detection strategies which informed the development of ACI items, were composed of three items each: supposed symptoms, exaggerated symptoms, symptom combinations, and selectivity of symptom report.Utility of the ADHD Credibility Index (ACI) in the detection of non-credible, self-reported symptoms of ADHD was dependent on the sample under study. The ACI detected instructed simulators at rates comparable to existing embedded validity indicators, particularly those described in early studies on the CII . ROC analysis indicated that neither of these infrequency indices, nor the CAARS DSM scales, performed significantly above chance. In light of the non-credible group’s small sample size, these results ought to be interpreted with utmost caution. Yet, they may underscore divergence of results provided by SVTs and PVTs  and Simulation (79%) Groups fell in between. Divergence most commonly resulted from respondents being identified as suspect by the CII but not the ACI, which detected 50% of participants whose CII scores fell above the cut-off value. Approximately half of those not identified by the ACI were simulators, illustrating the CII’s superior sensitivity to feigned instances of ADHD. However, 30% of respondents identified only by the CII were credible patients with ADHD and 11% were controls. Seeing that CII items stem from an instrument intended to measure symptoms of ADHD, this greater number of adults with the disorder being identified by the CII—compared to the ACI—may come to no surprise. Minor overlap between the CII and ACI is in line with findings suggesting that the CII and EI, too, each detect different subgroups of examinees  and therefore the largest difference between genuine cases of adult ADHD and simulators. Since items of this subscale tap complaints laypeople may erroneously associate with ADHD, this is consistent with previous studies recommending the use of stereotypes and misconceptions in the detection of feigned ADHD . This was due to a substantial number of participants with ADHD endorsing these items, suggesting that the presented combinations of symptoms were not sufficiently rare in our sample of genuine patients after all. Subscales examining exaggerated symptoms and selectivity of symptom complaints revealed small effects for the comparison of credible patients with ADHD and instructed simulators. Interpretation of these effect sizes requires caution, as data were non-normally distributed.Considering individual subscales rather than the ACI sum score, differences emerged between the four detection strategies which formed the ACI’s theoretical basis. Inquiries into supposed symptoms revealed a medium effect Below is the link to the electronic supplementary material.


The Secret Trail of Moon (TSTM) is a virtual reality serious video game that was designed for cognitive training related to core ADHD symptoms and executive dysfunction.Attention-deficit/hyperactivity disorder (ADHD) affects between 4% and 8% of children worldwide. The treatment of choice is multimodal treatment. Multimodal interventions for ADHD may be improved by incorporating new treatments, such as treatment via serious video games. We aimed to describe the development and usability of TSTM.The usability study included 37 children and adolescents who tested TSTM during the early usability stage (preinclusion) of a randomized controlled clinical trial for testing the effectiveness of TSTM. Chi-square tests were performed to compare patients with ADHD (ADHD combined subtype vs inattentive subtype) and to compare frequent and infrequent video game players in the second study. We used SPSS version 20 for Macintosh (IBM Corporation).A total of 31/37 (86%) and 30/37 (83%) of participants liked playing TSTM and wanted to continue playing TSTM, respectively. Further, 5/37 (14%) of participants reported that they experienced either perceived dizziness or virtual reality motion sickness. We found no statistically significant differences after comparing the ADHD combined subtype to the inattentive subtype and frequent video game players to infrequent video game players.Serious video games, such as TSTM, may complement the current multimodal approach for treating ADHD.ClinicalTrials.gov NCT04355065; https://clinicaltrials.gov/ct2/show/NCT04355065 Attention-deficit/hyperactivity disorder (ADHD) is the most common neurodevelopmental disorder of childhood and adolescence; it affects 4% to 8% of children worldwide . Apart fSome recent proposals include the use of board games, such as chess ; neurofeAnother problem is the lack of evidence regarding the transfer of improvements and benefits. In other words, it is not known whether improvements in video game performance would translate into improvements in other cognitive tasks in a subject's daily life. For instance, regular chess use has been demonstrated to transfer benefits to the educational domain  . HoweverPlan-It Commander—a serious game that was developed for children with ADHD [The development of any serious video game requires a series of stages ,25,26—(1ith ADHD . They inith ADHD . Afterwaith ADHD . They foith ADHD .The Secret Trail of Moon (TSTM)—a serious video game that was specifically created to train patients with ADHD and increase various cognitive abilities. This usability study allowed us to (1) obtain initial feedback from patients with ADHD; (2) detect bugs and integrate improvements; and (3) confirm that the use of our VR video game was attractive, was intuitive, and did not generate severe adverse effects in users.In addition to the development stages reported above, we present the third step (usability) of the development of The development stages of TSTM are shown in Information about the first stage of TSTM development can be found elsewhere . The maiTSTM is the result of interdisciplinary collaboration among health care professionals, researchers, serious video game consultants, and a single educational and therapeutic game development company. This collaboration made it possible to integrate several theoretical models of ADHD with clinical experience, the use of chess and VR by patients with ADHD, and other playful elements of video games that increase players’ motivation for engaging in therapy .TSTM is theoretically driven by (1) both the Barkley  and BrowThe Brown ADHD model postulates that the core deficit depends on executive dysfunction. In the Brown model, ADHD is considered to involve 6 major deficits in activation, focus, effort (motivation), emotion, memory, and action. In the Brown model, similar to the Barkley model, executive dysfunction is core to ADHD. However, both models have some differences . The BarThe TSTM prototype that was used in this usability study had 5 minigames; each minigame targeted specific cognitive abilities  and partAfter an initial design of TSTM was produced, we iteratively tested modified versions in order to detect and correct all bugs . FurtherIn conclusion, TSTM is a serious video game that is defined as either a game that was designed for a primary purpose other than entertainment  or a “coThe objective of our market study was to determine whether brain training via a serious video game, such as TSTM, would be of interest to mental health and education professionals. A survey that included the characteristics of TSTM and general questions about the game was designed. We used the Google Forms platform to disseminate Spanish and English versions of the survey through professional networks . A totalWe only analyzed the percentage of responses. No statistical test was needed.The survey was completed by 56 mental health (mostly psychologists and neuropsychologists) and education professionals , of which 71% (40/56) either treat and educate or have treated and educated people with ADHD. A total of 91% (51/56) and 87% (49/56) of the professionals thought that a serious video game such as TSTM could be useful and stated that they would use it as a therapy and educational tool, respectively .Responses about the characteristics of TSTM helped us to prioritize some development goals Some of the recommendations that were made were in line with literature that stressed the relevance of using attractive graphics or minigame mechanics with an increasing difficulty curve ,40.The second study included the first 40 consecutive patients with ADHD who were offered inclusion into a randomized controlled clinical trial (RCT) for testing the effectiveness of either TSTM or web-based chess . This RCThe patients included in this early usability stage tested the initial version of TSTM between December 6, 2019, and February 22, 2020, at preinclusion. In order to provide all available information before entering into the RCT, patients were offered the opportunity to test TSTM at preinclusion. Of the first 40 patients, 3 were excluded from the statistical analyses either because they did not complete the test or because the data provided were incomplete. The remaining 37 patients tested TSTM and eventually entered the study. However, not all patients were allocated to TSTM group, as randomization took place during the inclusion visit (day 0), and the data presented in this paper were recorded at preinclusion.The inclusion criteria were (1) an age of 12-22 years, (2) competency in the Spanish language, and (3) written informed consent. The exclusion criterion was epilepsy, since the use of VR is not recommended for individuals with epilepsy, as per the official recommendations of PlayStation VR . EthicalThe materials used for this study included the video game itself (TSTM), VR glasses, PlayStation 4 controllers (Sony Group Corporation), test kit consoles, monitoring screens, and headphones. The VR software runs on a PlayStation 4 (Sony Group Corporation) test device, which allows for the tracking of movement through the camera .Demographics, previous VR experience, and vision problems were noted by using an ad hoc questionnaire. The testing time was always monitored by a professional looking through a screen .Initially, participants attended a single session in which they tested the TSTM experience. Patients with ADHD were given the opportunity to test a single minigame. After passing the tutorial of the minigame to be tested, a higher level of difficulty was tested. Patients could test each minigame for as long as they liked . After testing a single minigame, patients rated their opinion about the minigame by using an ad hoc questionnaire, which included (1) 5 general questions about the TSTM experience and (2) 2 questions about possible adverse effects. Based on previous studies -46, we iParticipants were divided into the ADHD combined and inattentive subtypes because some authors consider these subtypes to be different disorders and believe that they should be treated separately. Thus, we feared that the different subtypes of ADHD would impact our usability measures. A descriptive analysis was performed by using the mean proportions (%) of participants’ ratings for categorical variables and means and SDs for numerical variables. Chi-square tests were performed to compare patients with ADHD (ADHD combined subtype vs inattentive subtype) and to compare frequent and infrequent video game players. We used SPSS version 20 for Macintosh (IBM Corporation).Kuburi: 22/22; Teka Teki: 23/23). With regard to the results for comprehensibility, the ease of play, and the ease of control of the PlayStation 4 controller, all proportions surpassed 60% (22/36). All minigames were easy to play, and only Teka Teki had an easy-to-play percentage of below 80% . Teka Teki was the only minigame in which the participants pointed out that it was difficult to achieve improvement after repeating the minigame. We found no statistically significant differences after comparing the ADHD combined subtype to the inattentive subtype and frequent video game players to infrequent video game players.Our study expands on current knowledge concerning the development of serious video games for treating patients with ADHD -28,33,48Teka Teki and Kuburi) were the most dynamic and interactive minigames. The areas for improvement in Smasher appear to be making it a little bit easier to understand and extending the play time. Kuburi was the minigame with the highest usability ratings across all parameters. Enigma was the minigame that was the most difficult for users to comprehend. Thus, we may have made the graphics too attractive or enhanced minigame mechanics with an increasing difficulty curve [Teka Teki was the most difficult minigame to play and the only minigame in which participants found it difficult to achieve improvement after repeating the minigame. As such, this minigame may need some improvement, such as using a less stringent difficulty curve and thus preventing a potential decline in initial motivation [Our results suggest that TSTM was fun, was understandable, was easy to play, was intuitive, was easy to master, used enjoyable graphics, and was of adequate duration for most participants. However, there were some interesting differences between each minigame that may suggest that certain components of TSTM need more improvement. The most enjoyable minigames  of our participants reported feeling good or very good after testing TSTM. Furthermore, our results are comparable to those reported in the Plan-It Commander study with regard to players’ motivation to play the game again and their opinions of the game [Compared to a similar study, our study yielded better satisfaction percentages than those reported by the users of ommander . For insthe game . Finallythe game -46.Our usability study presents the following limitations. First, not all of the participants tested the same versions of each minigame during the usability phase, although the changes made were kept at a minimum. We used the preinclusion period of the RCT to fix the bugs detected by participants, and we made some improvements based on their ongoing suggestions in an iterative, continuous way. Thus, the data reported in this paper were the data collected during the preinclusion period. However, after the preinclusion period, all included participants in the RCT eventually tested the same TSTM version. Second, our questionnaire was not validated but was based on questionnaires that were used in similar studies . FinallyPlan-It Commander) group to a treatment-as-usual crossover group (the intervention was used as an adjunct treatment for children with ADHD), the 10-week serious game intervention proved to be a more effective strategy [Serious video games and VR are new technologies that can be used as therapeutic tools for the treatment of mental disorders ,50. Compstrategy . Howeverstrategy ,57; the TSTM—a VR serious video game that was designed for patients with ADHD—was fun, was intuitive, and displayed a favorable profile of side effects that were in line with those reported in literature -46. Addi