Many of the algorithms and software suites commonly applied for in silico druggability assessment are complex, technically challenging and not always user-friendly. Here we applied the intuitive open access servers of DoGSite, FTMap and CryptoSite to comprehensively predict ligand binding pockets, druggability scores and conformationally active regions of the NUDIX protein family. In parallel we analyzed potential ligand binding sites, their druggability and pocket parameter using Schrödinger's SiteMap. Then an in silico docking cascade of a subset of the ZINC FragNow library using the Glide docking program was performed to assess identified pockets for large-scale small-molecule binding. Subsequently, this initial dual ranking of druggable sites within the NUDIX protein family was benchmarked against experimental hit rates obtained both in-house and by others from traditional biochemical and fragment screening campaigns. The observed correlation suggests that the presented user-friendly workflow of a dual parallel in silico druggability assessment is applicable as a standalone method for decision on target prioritization and exclusion in future screening campaigns.Computational chemistry has now been widely accepted as a useful tool for shortening lead times in early drug discovery. When selecting new potential drug targets, it is important to assess the likelihood of finding suitable starting points for lead generation before pursuing costly high-throughput screening campaigns. By exploiting available high-resolution crystal structures, an  Structural and domain analysis revealed three major groups and one outlier, NUDT22, mostly based on their already reported activity against substrate classes such as diphosphoinositol polyphosphates  hydrolase protein family was recently comprehensively and exhaustively reviewed by Carreras-Puigvert et al.  NUDIX pr,Besides NUDT1, a series of potent, drug-like NUDT5 inhibitors have been described by Page and coworkers  The structures were then prepared using the Protein Preparation Wizard as implemented in the Schrödinger Suite. Briefly, raw PDB structures were processed by automatically assigning bond orders, adding hydrogens, creating zero-order bonds to metals, converting selenomethionine to methionine, adding missing side-chains, creating possible disulfide bridges, deleting waters beyond 5.0 Å of hetero groups (if present), and generating hetero protonation states at pH 7.0. Residues with alternate positions were locked in the conformations with the highest average occupancy. Small ligands and metal ions originating from crystallization buffer were removed. The hydrogen bonding networks were optimized automatically, by sampling water orientations and optimization of hydroxyls, Asn, Gln, and His residue states using ProtAssign. Any remaining water molecules were subsequently removed. A restrained minimization was then performed using the OPLS3e force field, until an RMSD convergence of 0.30 Å was reached for the heavy atoms. Finally, the minimized NUDIX structures were aligned to the structure of NUDT1 (3Q93) with respect to the backbone atoms of the A chain.The protein structures as prepared above were exported as PDB files, uploaded to the DoGSite server and assessed for binding sites and their corresponding DrugScores according to the published protocol  were used. Site maps at 4 Å or more from the nearest site points were cropped. Clustering of the SiteMap parameters was performed using the heatmaply library in R1) Fragment subset selection: a subset of the ZINC Frags Now set  Ligand preparation: the selected fragment subset was then prepared for docking using LigPrep (Schrödinger): the OPLS3e force field was used for minimizations; possible ionization states at pH 7.0 ± 2.0 were generated using Epik  Receptor grid generation: Glide docking grids  Virtual screening: The Virtual Screening Workflow as implemented in Schrödinger Suite was used for docking, scoring, and ranking of the top-1,000 fragments against the sites with the highest Dscore as determined by SiteMap. The workflow comprised a cascade of docking steps with increased accuracy (Glide HTVS → SP → XP), where the top-10% ranked ligands are passed on to the next step. After Glide XP docking the top-1,000 ranked fragments were retained for druggability assessment based on their combined docking scores.E. coli and subsequent validation of coupled enzymatic assays based on cognate substrates for each of these [Ap4A for NUDT2, dGTP for NUDT15 and ADP for NUDT16  and positive controls (no enzyme or inhibitor at concentration that gives 100% inhibition). These are located in columns 23 and 24 of the 384-well plates and they are used to normalize the response in each well-containing library compounds to a % inhibition value. Hit limits are defined based on the average plus three standard deviations of the response for all library compounds and hit rates are provided as the percentage of library compounds above this limit. The malachite green assay has been extensively used for screening purposes in our lab as it is associated with low interference rates, as evidenced by the lack of common hits appearing in screens of NUDT1  and NUDT18 , individually score a high DrugScore around 0.80. Furthermore, it can be observed that several members, including NUDT6 , NUDT7 , NUDT9 , NUDT17 , NUDT12 , and NUDT22 similar in terms of their active site properties rather than based on sequence (dis)similarities . Full-leNUDT15 and NUDT18 have been hypothesized to be able to act as back-up enzymes for NUDT1 due to their overlapping substrate specificities. Comparison of their SiteMap parameter profiles shows clear differences despite NUDT1 and NUDT15 being among the NUDIX members with highest Dscores. The active site of NUDT15 is somewhat smaller and more enclosed than for NUDT1 due to an inward movement of a helix  can be observed , NUDT5 , NUDT7 , NUDT15 , NUDT17 , and NUDT22  and disfavors NUDT3 , NUDT4 , NUDT10 , and PTP1B . With regard to their returned median fragment docking scores, pocket polarity might correlate with either higher or lower scores . In fact, 73 fragments bound to 6 or more targets . TL is an employee of AstraZeneca, but performed all experimental work associated with this publication while at Chemical Biology Consortium Sweden. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Understanding household-level transmission pathways of fecal pathogens can provide insight for developing effective strategies to reduce diarrheal illness in low- and middle-income countries. We applied whole bacterial community analysis to investigate pathways of bacterial transmission in 50 rural Bangladeshi households. SourceTracker was used to quantify the shared microbial community in household reservoirs  and estimate the percentage of fecal-associated bacteria from child and mothers’ feces in these reservoirs. Among the reservoirs studied, most bacterial transfer occurred between mothers’ and children’s hands and between mothers’ hands and stored water. The relative percentage of human fecal-associated bacteria in all household reservoirs was low. We also quantified the number of identical amplicon sequence variants within and between individual households to assess bacterial community exchange in the domestic environment. Intra-household sharing of bacteria between mothers’ and children’s hands and between hands and soil was significantly greater than inter-household sharing.  •16S rRNA gene sequencing was used to examine bacterial communities in households.•Low levels of fecal contamination were found on hands, in soil, and stored water.•High proportion of bacteria was shared between mothers and their children. WASHWater, sanitation, and hygieneASVAmplicon sequence variantLMICLow-and middle-income countrySSoilSWStored waterCHChild handsMHMother handsCFChild fecesMFMother feces1Enteric pathogens are transmitted via the fecal-oral route through a variety of pathways in the environment. Pathogens can move through environmental reservoirs including flies, fomites, hands, soil, food, and water. Providing sufficient quantities of water, adequate drinking water treatment, sanitation, and hygiene (WASH) are the main strategies to block pathogen transmission pathways and reduce the occurrence of diarrheal illnesses. Recent field trials have investigated the impact of these interventions on diarrhea in children and found limited reductions (even with high levels of compliance) and no additive benefit of multiple interventions . ImproviE. coli and microbial source tracking markers, in household reservoirs such as soil, hands, stored drinking water, and children’s toys , has been used to explore sources of fecal contamination in the natural environment as well as in the built environment by characterizing the shared microbiomes in homes, kitchens, hospitals, and restrooms . SourceTThe goal of this study was to apply bacterial community analysis to elucidate pathways of enteric pathogen transmission in 50 rural Bangladeshi households. Specifically, we aimed to: 1) quantify the overlap in amplicon sequencing variants within and between individual households to distinguish intra-household bacterial community exchange; 2) quantify the shared microbial community in stored drinking water, soil, and child and mothers’ hands using SourceTracker; 3) estimate the percentage of human fecal-associated bacteria from child and mothers’ feces in household reservoirs, using SourceTracker, in an attempt to overcome the limitation of low host specificity exhibited by the single target markers in rural Bangladesh ; and 4) 22.1For this analysis, we had the opportunity to combine and sequence samples obtained from two studies conducted under the WASH Benefits randomized controlled trial in rural Bangladesh, which measured the impact of water, sanitation, hygiene, and nutrition interventions on child health outcomes . The env2 stencil from an area as close to the house entrance as possible. Stored water samples were collected by asking mothers to provide a glass of water as they would give to their child under five. Samples were transported to the icddr,b field laboratory on ice and were processed within 12 h. At the field laboratory, 50 mL of hand rinse sample and up to 500 mL of stored water was filtered through a 0.45 μm HA filter . Filters were treated with 0.5 mL of RNAlater and stored at −80 °C. Filter and soil samples were transported to UC Berkeley for DNA extraction and 16S rRNA gene sequencing.50 control arm households, sampled as part of the environmental analysis, were selected for this study. Households were chosen based on availability of all environmental sample types (child hands (CH), mother hands (MH), stored water (SW), and soil (S)) collected at the same visit . DetaileAn additional 25 soil samples were selected from the control households for the source tracking soil library. To attempt to decouple the contributions from soil and feces, soil samples for the library were selected based on the absence of the human (HumM2) and animal (BacCow) fecal markers, as determined by qPCR. However, HumM2 had a relatively low sensitivity and specificity for human feces in rural Bangladesh, and it is possible human fecal contamination was missed by this marker . Soil sa2.225 maternal fecal (MF) and 25 child fecal (CF) samples were obtained from households enrolled in the control and combined water, sanitation, and hygiene arms of WASH Benefits. Maternal and child fecal samples were not from the same households as those used for environmental analysis because fecal samples obtained for the enteric infection study  n. d. sa2.3Library preparation and sequencing was performed at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley. The V4 region of the 16S rRNA gene was amplified using 515f and 806r primers. Samples were pooled and sequenced on two MiSeq runs, yielding paired-end 250 bp reads. A mock community DNA standard  was included on each MiSeq run. Approximately 13 million total reads were obtained from run one and 14 million reads from run two. An average of 50,460 reads per sample were obtained from child hands, 50,595 from mother hands, 87,160 from stored water, 89,514 from soil, 112,142 from child feces, and 117,675 from mother feces.2.4Forward and reverse reads were processed using the DADA2 pipeline . Reads wEscherichia coli, Aeromonas hydrophila, Campylobacter jejuni, Vibrio cholera, and Salmonella enterica)  . We inclPseudomonas syringae pv. phaseolicola (pph6) to estimate extraction efficiency in previous work  in R (version 3.5.0) . A subseous work . This ASous work . Beta dious work . For allOverlapping ASVs  within the same household, versus between different households, were determined by identifying the number of identical ASVs present in each sample type within the same household and in different households. To determine if the mean number of ASVs overlapping within and between households was significantly different, we used a bootstrap method . The aveTo estimate the association between bacterial communities in different reservoirs and feces, we used SourceTracker2 in python (version 3.7.0) . SourceTThe source tracking analysis was validated using two approaches. In both, different percentages of DNA from samples of sources  and sinks  were prepared in the laboratory, and then compared to the percentages estimated using SourceTracker. All source and sink samples were composites, which were prepared by pooling equal masses of DNA extract from five individual samples of the same sample type. In validation approach one, DNA extract from each source  was spiked into DNA extract for each sink  to achieve DNA concentration percentages of 10% source/90% sink, 1% source/99% sink and 0.5% source/99.5% sink. In approach two, child feces, mother feces, and soil source samples were combined in different DNA concentration percentages from 0 to 80%. All validation combinations are shown in 33.1The relative abundance of the ZymoBIOMICS mock DNA community constituents was within 3 ± 2% between the two runs . The eig3.2There was good agreement between estimated source contributions and SourceTracker predicted contributions . SourceT3.32 = 0.27, p = 0.001). While samples appear to group based on sample type, it should be noted that there were some soil, stored water, and hand samples that cluster together and a low percentage of the variation was explained by the two axes (21%). In fecal samples, there was a significant difference in the communities between mothers and children . PERMANOVA tests were also performed using the Aitchison distance as suggested by 2 values were similar and the difference in bacterial community by sample type was also statistically significant in these models.Sample type explained a significant difference in the bacterial community  , compared to the hands of mothers in other households (mean = 98) . The low3.5Mother and child hands had the highest estimated percentage of associated bacteria of all reservoirs based on SourceTracker results . On chilIn stored water, the estimated percentage of ASVs associated with mother and child feces was less than 0.5%. A greater percentage of the bacterial community was associated with hands , with a significantly greater percentage associated with mother hands (p < 0.001). On average 9.6 [5.9–13.3]% of the microbial community was related to soil, which was significantly greater than feces (p < 0.001 for both). However, the estimated percentage of ASVs associated with mother hands was significantly greater than the estimated percentage associated with soil (p = 0.01). In soil, less than 0.5% of the bacteria were human fecal-associated. When low abundant taxa were removed, the SourceTracker results were similar on mother and child hands . In stor3.6Sphingomonadaceae was one of the most abundant families on hands, in soil, and in stored water . The relative abundance of these potential pathogens was low in all sample types (<1%). We also assigned taxonomy using the RDP database . This finding was unexpected given that disposal of child feces in Bangladesh typically involves caretaker handling of feces , and it Rhodobacteraceae and Nocardioidaceae. The most abundant bacteria families in our study on mother hands were skin and environmental- associated Moraxellaceae followed by skin-associated Micrococcaceae.The importance of the hand-soil pathway is also supported by the overlapping ASV analysis; more bacteria on mother and child hands were identical to bacteria in the soil in their own household compared to soil from other households. Soil has been shown to be contaminated with many different pathogens in LMICs , includikolshi  that can be covered or uncovered. Water is then dispensed by pouring directly into a vessel for drinking or into an intermediary container, such as a pitcher, or by reaching into the container with a cup, which could result in the contamination of source water by mother hands. However, there was only a borderline significant difference in the number of identical bacteria on hands and in stored water within the same household compared to stored water from other households .The highest percentage of bacteria in the stored water microbial community was attributed to bacteria on mother hands (aside from the unknown category). In Bangladesh, water is typically collected from shallow tubewells and stored in a useholds . A relatce water , althougce water . The stuHand to hand transmission between mother and child is an important pathway for the exchange of bacteria. Looking at the overall microbial community composition, mother and child hands were very similar , Fig. 3,Campylobacter which was not a pathogen investigated using targeted analysis (qPCR and PCR) in related work  or qPCR. However, in order to identify pathogens that are present in low relative abundance in environmental samples, considerably higher sequencing depth is required using metagenomics compared to amplicon methods, significantly increasing cost. Additionally, assigning species relies on the accuracy of existing databases. We found no difference in the taxonomy assigned to potential pathogen ASVs at the genus level between Silva and RDP, but a previous study estimated annotation error rates in these databases to be 17% and 10%, respectively (Of the potential pathogens we identified, almost half of the ASVs were from the genera ted work , but hasted work . In resoectively . AnotherWhile we observed quantitative differences in the contribution of varying sources, further validation of this approach is needed, especially for sources with estimated low percent contributions. Unlike previous work that used SourceTracker to identify sources of contamination based on threshold values, we have included all SourceTracker results in our analysis and have avoided using thresholds for assigning a source. In Bauza et al. a 1% threshold was used as the cut off value to consider samples positive for source fecal contamination or if the source was positive in at least 3 of 5 runs with an average relative standard deviation less than 100% . AlthougA limitation of this study design is that it relied on repurposed samples originally collected for other related work , 2019 anBacterial community analysis is a promising approach for understanding pathways of enteric pathogen transmission. Although we show relatively low proportions of human fecal bacteria in the bacterial community in environmental reservoirs, low levels of fecal contamination can still transmit pathogens. Nonetheless, we were able to identify pathways of bacteria transfer between mother and child hands, soil and child hands, and soil and mother hands at the household level. This non-targeted approach may be more appropriate to use in LMIC settings where sensitivity and specificity of source tracking markers is impacted by the potential sharing of microbial communities between humans and animals. Even though sharing of microbial communities between humans and animals is also a limitation in differentiating sources using SourceTracker, the use of multiple markers as indicators of fecal contamination could be more robust to similarity in source microbial communities than single targets, and further exploration is warranted.5•Only a small fraction of the bacterial community in household reservoirs was human fecal-associated, although low levels of fecal contamination can still pose a health risk.•The percentage of fecal-associated ASVs on hands in rural Bangladesh was similar to the percentage of fecal-associated bacteria found on hands in the US in a previously published study.•Mother and child hands had the highest percentage of associated bacteria and significantly more bacteria were identical between mother and child hands within the same household, compared to those from other households.•In stored water, the highest percentage of bacteria from a known source was associated with mother hands.•The percentage of ASVs from unknown sources was high and future work should include more household source types.Raw sequence reads for this study were deposited into the sequence read archives under BioProject PRJNA604825.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


BackgroundSpastic triplegia is a recognized subtype of cerebral palsy (CP). In the course of treating spastic triplegic children with selective dorsal rhizotomy (SDR), we found that some children who had “minimal or mild involvement” in the stronger arm improved strikingly after undergoing SDR. Some of them became independent ambulators, which was an outcome that is not usually achieved in spastic quadriplegic children. However, the literature currently contains no data on the natural clinical course and the effects of CP interventions on spastic triplegia.ObjectivesOur aim was to elucidate the clinical characteristics of spastic triplegia and the effects of SDR on functional outcomes and the quality of life after childhood SDR.MethodsThe Institutional Review Board of the Washington University School of Medicine approved this quality of life survey (number: 201908177). The subjects of this study were children and adults  with spastic triplegic CP who had undergone SDR  between 2003 and 2018 at the St. Louis Children’s Hospital. The follow-up period ranged from 1-16 years (mean: 6.0 ± 4.3 years). The study included a 76-patient cohort selected from a total of 253 spastic triplegic CP patients who had undergone SDR. All 253 patients were contacted via email or postal mail soliciting their participation in the study including the survey. The cohort included all patients who responded. The survey included questions on demographic information, quality of life, perceptions of health and the SDR procedure, motor and ambulatory functions, braces and orthotics, pain issues, side effects of SDR, and post-SDR treatment.ResultsThirteen patients had presented with scissored gait, and these patients had undergone partial ventral rhizotomy (PVR) of L1-3 ventral roots immediately after the completion of SDR. Of note, 91% of 76 patients reported that SDR improved their quality of life, and 93% would recommend the procedure to other patients. After SDR, 21 more patients were able to run, 14 more played recreational sports, and 18 more could walk without using walking aids. Sixteen fewer patients used a wheelchair for long-distance walking and in crowds; 37 and 32 patients reported an improvement in the more affected arm and hand, respectively. Sixty-eight patients were able to regularly strengthened their muscles at least once a week, and 60 patients regularly stretched their legs. However, 53 patients required assistance with bathing or showering, 50 with getting dressed, and 56 with grooming or hygiene. Forty-eight patients had orthopedic surgery after SDR. Percutaneous hamstring-lengthening was the most common type of orthopedic surgery performed. Three of 13 patients who received PVR and SDR required adductor release. Six patients used medications for spasticity or dystonia. No late side effects of SDR were observed.ConclusionsOur report elucidates the clinical features of spastic triplegia before and after SDR. A distinct clinical feature was the wide variation in ambulatory functions, ranging from total independent walking to wheelchair mobility. The vast majority of patients felt that SDR improved their motor functions and quality of life. PVR also resulted in favorable outcomes, with only three of 13 patients requiring additional adductor release surgery. There were no late complications related to SDR surgery. Spastic triplegia is a recognized subtype of spastic cerebral palsy (CP) . The curWe have observed that none of the spastic quadriplegic patients become independent ambulators after undergoing selective dorsal rhizotomy (SDR). The quadriplegic patients need a wheelchair or an assistive device to walk even after undergoing SDR. By contrast, children with spastic triplegia who have “minimal or mild involvement” of the stronger arm show markedly improved motor functions after SDR. Some of them even progress to walk independently. The current literature includes no data on the natural course of spastic triplegia and outcomes after any CP therapeutic intervention. In the present study, we examined the outcome of spastic triplegia patients in the areas of gross motor functions, arm improvement, living status, improvement of daily life activities, perceptions of SDR by parents and patients, and orthopedic surgery and other treatments after SDR.The Institutional Review Board of the Washington University School of Medicine approved this quality of life survey (No. 201908177). We obtained consent from patients directly or from their guardians. The subjects of this study were children and adults  with spastic triplegic CP who had undergone SDR  between 2003 and 2018 at the St. Louis Children’s Hospital. We gathered contact information from emails, mailing addresses, and phone numbers recorded in our clinic’s database and medical records. The study survey was sent out to potential participants electronically via email or physically via postal mail to soliciting their participation.The survey included questions on demographic information, quality of life, perception of health, the SDR procedure, motor and ambulatory functions, braces and orthotics, pain issues, side effects of SDR, and post-SDR treatment. Demographic information included the date of birth, current living situation, student status, and employment. Questions about bladder function, loss of sensation, and back pain were included to assess the side effects of SDR. Participants who indicated a sensory loss in the lower limbs were asked to indicate the location of the numbness. Patients were asked the frequency, level , and location of the body pain. Participants were also inquired about scoliosis, post-SDR orthopedic interventions, and medications.A five-point scale from poor to excellent was utilized to evaluate the perception of one’s personal health, as seen in Question 1 of the SF-36 health survey . Yes/no/Patients were given the option to answer “Yes” or “No” to questions about motor function, including running, walking, and sitting. These questions were based on criteria determining the Gross Motor Function Classification (GMFCS) level of the patient . RegardiStudy cohortInitially, our target population consisted of 253 spastic triplegic CP patients who had undergone SDR. The definition of spastic triplegia covered the involvement of both lower limbs and one upper limb. The other upper limb was understood to be minimally or mildly involved. The research team obtained the contact information of the patients from the database of the Center for Cerebral Palsy Spasticity at the St. Louis Children’s Hospital. Thirty-seven patients did not have updated emails or postal addresses and could not be contacted, leaving 216 potential participants. Seventy-eight patients responded. Two of the 78 patients who responded declined to participate. Thus, we eventually had a 76-patient cohort for our study, accounting for 35% of the initially considered participants.All 76 patients had undergone the SDR procedure as described previously . The ageGross motor functions and daily physical activitiesFifty-eight patients still required assistance in daily activities even after SDR. The patients required the most assistance with activities related to grooming and hygiene; 53 patients required assistance with bathing or showering, 50 with getting dressed, and 56 with grooming or hygiene. However, only 21 patients required help with eating . Fourteen more patients enjoyed recreational sports, 21 more could run and jump, and 18 more could walk without using walking aids. Patients requiring extra body or trunk support to improve arm and hand function went down from 58% (44 patients) to 18% (14 patients) after SDR. Fewer patients relied on wheelchairs for long distances and crowds (down from 61 patients to 45 patients). However, there was no change in the number of patients (11) who relied on powered wheelchairs for self-mobility  believed that SDR had improved their quality of life, and 71 patients (93%) said they would recommend the procedure to help other patients Table .Postoperative painThirty-seven patients reported having body pain post-SDR. Two patients reported constant pain, 45 occasional pain, and 36 reported no pain. Three patients did not have a response to body pain frequency. On a numerical scale of 1-10, with 1 being almost no pain and 10 being the worst pain imaginable, patients' responses ranged from 1 to 9 (mean: 3.3 ± 1.8). One patient answered with 9 and reported pain only in the ankle and foot. Others who reported a pain level of 5 and higher mostly reported pain in the general leg area and back. One patient reported a complete loss of sensation in any part of the legs. Two patients reported experiencing back pain directly related to the SDR. Four patients received treatment from a physician for their back pain, and no patient reported taking medication or seeking surgical treatment for back pain . A high proportion of patients use ankle-foot orthosis . Others use supramalleolar orthosis , shoe inserts (10 patients), and others (four patients). Other braces and orthotics used include, but are not limited to, night orthotics, Bioness , and immobilizers Table .Perceptions of health Almost all patients (74 patients) reported that their health was good or better. Twenty-one patients reported excellent health, 34 very good, 19 good, two fair, and none reported poor health Table .Living situation, education, and employmentSixty-seven patients reported living with their parents, six with a significant other, two with a roommate, and one reported living alone. Sixty-one patients were full-time students, and 10 were not students. Thirteen patients were employed at the time of the survey; two of the employed patients were working full-time, 10 were working part-time, and one patient did not specify the employment status Table .The present study was a retrospective analysis of survey responses from 76 patients out of 216 35%) potential participants. Thus the study has some limitations [% potentiIt is remarkable that in our series of spastic triplegic patients, the preoperative walking ability varied from complete independent walking and playing recreational sports to a total reliance on wheelchairs. By contrast, none of our spastic quadriplegic patients could walk independently after SDR. Elaboration on a few clinical insights is worthwhile. First, after SDR, virtually all mobility-related activities and the quality of walking improved Table . For patIn young children under five years of age, the scissoring itself prevents progression in gait development, and children cannot learn to walk. SDR alone cannot resolve scissoring caused by adductor contractures. We treated the scissored gait with PVR. The surgery entails dividing 30-50% of L1-L3 ventral roots at the time of SDR. Electrical stimulation of the ventral roots is not useful because of the overlapped innervation of the muscles in the lower limb. The level of ventral roots is identified by anatomy and muscle contraction in response to direct tap on the ventral root ,9. In ouThirteen patients (17%) had presented with scissored gait before the operation and all of them had gone on to receive PVR after SDR. PVR was successful in 10 of the 13 patients, and no adductor release was necessary after the procedure. The remaining three patients had recurrent scissoring and required percutaneous adductor lengthening. PVR is our first choice for the treatment of scissored gait due to the adductor contracture. They stretch the adductors daily while the adductor weakness after PVR improves for four months. In our experience, PVR seems more successful in children between the ages of two to age than in older children.Significantly, 48 patients required orthopedic surgery after SDR. The correct orthopedic surgery following SDR complements SDR . OrthopeIt is gratifying to note that that 91% (69 of 76 triplegic patients) of survey participants felt that SDR had improved their quality of life, and 93% (71 patients) said they would recommend SDR to other patients. None of the patients who did not participate in the survey reported that SDR was a total failure in all aspects of motor movements, daily life activities, independence, and confidence. It is encouraging that 74 of 76 patients reported good or better health even though some of them reported mild body pain. Another encouraging fact was that 80% (61 patients) of the participants were full-time students.Our study elucidated and analyzed the clinical features of spastic triplegia. A distinct clinical feature of spastic triplegia is the wide variation in ambulatory functions, ranging from total independent walking to wheelchair mobility. The vast majority of our patients felt that SDR had improved their motor functions and quality of life. The outcomes of PVR procedures were also favorable. There were no late complications related to SDR surgery.


Nothofagus pumilio is one of their most ecologically important tree species . It presents great adaptability and a wide distribution range, making it a suitable model for predicting the performance of trees facing global climate change. N. pumilio forests are increasingly threatened by extreme climatic events and anthropogenic activities. This study aims to identify priority conservation areas and Genetic Zones (GZs) for N. pumilio, promoting the implementation of specific practices to ensure its management and long-term preservation. Thirty-five populations (965 trees) sampled across its distribution  were genotyped with SSRs, and geographical patterns of genetic variation were identified using Bayesian approaches. The phylogeographic patterns of the species and geomorphological history of the region were also considered. Six priority conservation areas were identified, which hold high allelic richness and/or exclusive allelic variants. Eighteen GZs were delineated based on the genetic structure of this species, and maps showing their distributional range were drawn up. Overall, this study defines management units based on genetic data for N. pumilio for the first time, which will facilitate the establishment of sustainable practices and highlight priorities for investment of conservation funding.Patagonian forests are the southernmost temperate forests in the world, and  The biological value of these forests lies partly in their high level of endemism and frequency of taxonomically isolated genera, despite low species richness in comparison with other South American forests1. Together with some conifer species, southern beeches of the Nothofagus genus constitute the main components of Andean Forests, which are recognized as providing a wide range of ecological, economic, and social benefits and services. Therefore, the management of these native forests is required in order to maintain the ecological dynamics of this unique ecosystem. Several studies have documented the role of climatic variability during the Quaternary in shaping the current distribution of Patagonian forests4. In addition, extreme climatic events have recently been suggested as one of the causal agents of current forest decline in Northern Patagonia5, e.g. severe droughts occurring after a wet period triggered N. pumilio decline6. In this regard, an increase in mean maximum summer temperature, overall warming, and a marked decrease in annual precipitation were detected in this region throughout the twentieth century8, threatening the persistence of these forests. Besides, anthropogenic and natural causes such as logging and more frequent fires10 also endanger these forests.Patagonian forests represent the most southern temperate forests in the world, and were isolated from other continental forests approximately 40–2.5 million years before presentNothofagus pumilio, commonly called lenga, is one of the most important species of Patagonian forests, since it has the largest distribution area of all native trees in the region, covering about 1.636 million hectares and occupying a range of about 20° latitude (from 36°S to 55°S) in Argentina. This species has a similar distributional range to the west of the Andes mountain range, in Chile11. Moreover, it occurs in heterogeneous environments along altitudinal, latitudinal, and longitudinal gradients that imply diverse and contrasting thermal, photoperiodic, and precipitation conditions, respectively. N. pumilio shows great adaptation or plasticity to extreme and contrasting environments, since it grows close to the timberline (cold-tolerant species) and thrives in a wide range of precipitation levels 12. In addition, several phenotypic variations have been described for this species across environmental clines; e.g., bud phenology variation and seedling growth associated with altitude14, and wood density related to soil moisture gradients15. Since N. pumilio exhibits great adaptability, it serves as a suitable model for prediction of the performance and fitness of trees to cope with global climate change in the southern hemisphere. Consequently, a research is currently being undertaken to determine whether local adaptation of this Southern beech evolved in a convergent way with respect to European tree species that are also distributed along broad environmental gradients . Natural hybridization between N. pumilio and N. antarctica has been observed in sympatric populations17 and it has been suggested that the latter acts predominantly as pollen receptor18. The introgression of the chloroplast genome from N. pumilio to N. antarctica would have occurred as a consequence of repeated backcrosses between hybrids and parents, and the former is clearly distinguished at the nuclear level19.Austrocedrus chilensis21, Nothofagus nervosa and Nothofagus obliqua23. According to the National Law N° 26331 on Minimum Requirements for Environmental Protection of Native Forests, most of the Argentinean N. pumilio forests (up to 97%) were included in areas of high (38%) or moderate (59.1%) conservation value24. Therefore, forest management is highly encouraged for this species, particularly the identification of priority conservation areas, in order to ensure compliance with this law and their long-term preservation. The identification of Genetic Zones (GZs) is one alternative of OGMU delineation. GZs are genetically homogeneous regions within which seeds can be transferred with low risk of causing change in the original genetic structure26. A requirement for the identification of GZs is the characterization of the population structure of a species, resulting from genetic signatures of evolutionary history, which can be assessed by neutral molecular markers. A second, well-known alternative for OGMU delineation is the definition of Provenance Regions (PRs), which aims to ensure the safe transference of propagation material without risk of maladaptation since it is based on variation in adaptive characters27. In addition, assessment of species’ genetic diversity with molecular markers could facilitate the identification of priority conservation areas29. Several authors have suggested that areas containing high allelic richness and/or exclusive allelic variants are meaningful for conservation purposes32, particularly because they play a key role as genetic diversity reservoirs or sources of locally-adapted genotypes.Government directives regulating the criteria and indicators for defining management units, such as Conservation Units (CU), became important worldwide during the last decades of the twentieth century. Appropriate management of native resources depends on understanding the nature and distribution of genetic diversity among and within species, and highlighting how this genetic variation is structured across their distribution range. Since most native forests are widely distributed and composed of hundreds of natural populations, management of their genetic resources cannot be based on single-population units. Thus, the establishment of operational genetic management units (OGMUs) is necessary for implementation of specific practices arising from conservation or restoration programs. In Argentina, OGMUs were delineated in three native species based on genetic criteria: N. pumilio forests. Genetically homogeneous regions  were first delineated by assessing the neutral variation of the target species across its natural distribution range in Argentina. Based on this, geographical patterns of N. pumilio genetic variation were recognized. In addition, areas holding high allelic richness and/or exclusive allelic variants were identified. Previous information from N. pumilio phylogeographic patterns33 was considered and new information from SSR markers was added.The aim of this work is to identify priority conservation areas in 34, due to its usefulness in determining the persistence and adaptive potential of a species. Therefore, indicators of genetic diversity should be considered when developing criteria and guidelines for forest management37. A set of genetic parameters was used here in order to characterize the N. pumilio populations . In particular, HE Nei for Tr population (0.757) was significantly higher than the HE Nei obtained for PG (0.581), Pi (0.573), BL (0.537), and HP (0.398) populations. In addition, both BL and HP exhibited the lowest heterozygosity values, suggesting that they hold lower genetic diversity than the other populations analyzed.Significant differences in unbiased heterozygosity were detected among populations using Wilcoxon’s test . Since this population is isolated from the main forest masses (western side of the species’ distribution) and located in an area that was covered by an ice field at the Last Glacial Maximum (LGM), we hypothesize that few individuals contributed to the recolonization process. In addition, a population bottleneck was identified in Sierra Colorada (SC), since it showed significant heterozygosis excess under both Infinite Allele Model -IAM and Two Phase Model -TPM mutation models .Inbreeding and bottlenecks are demographic events that shape population genetic variation, specifically, reducing allelic variants. Signs of inbreeding were detected in 12 populations  method . The indexes did not differ substantially, indicating that null alleles did not strongly bias the FST value. At the same time, the standardized Hedrick’s G′STH  was 0.201. These differentiation coefficients (FST and GST) were similar to those previously reported in other Nothofagus species such as N. nervosa (0.061) and N. obliqua (0.049)22.The differentiation coefficient for N. pumilio populations19. Based on these results, an estimation of population structure was carried out, implementing spatial clustering methods as recommended by Perez et al.38. In contrast, the genetic and geographic distances do correlate with each other when using maternally inherited cpDNA markers33. In this regard, it was suggested that the pollen dispersal capacity of N. pumilio greatly exceeds that of seed dispersal (75–100 times greater)39, as in other wind-pollinated trees41, which could partly explain the differences found between cpDNA and SSR results from IBD analyses.An isolation-by-distance (IBD) pattern was not supported by the Mantel test and the Spatial Genetic Structure (SGS) test  and Huemules (42° 50′S) in N. pumilio forests, since all individuals analyzed from these two sites exhibited either northern or southern haplotypes, respectively. The evolutionary significance of this lineage segregation prompted us to consider these findings as the first division criterion for exploration of the genetic structure of Argentinean N. pumilio forests located to the north and south of these boundaries  was reported as a putative relictual zone33 since it exhibits high genetic diversity in plants  and animals .From the genetic structure analysis (BAPS) of the ‘north’ subset, four main clusters were identified which grouped the populations with an adjustment probability of 0.941  were analyzed separately , other division criteria were also considered in defining the optimum number of clusters. A particular topographic configuration occurs at 44°S where there are changes in the orientation of the watersheds (north–south oriented mountain chains between 42°S and 44°S shift afterward to a west–east orientation). This feature contributed to different ice mass development during the glacial periodely Fig.  IIa. In 49. In addition, palynological records suggest a different paleoclimatic history for the area south of 50°S, which shaped the forest expansion in a different way  in southern Patagonia and Tierra del Fuego during the postglacial period3. Furthermore, a particular recolonization process was suggested for Tierra del Fuego Island, mainly led by trees from local periglacial refugia50. Currently, the N. pumilio Argentinean forest south of 44°S is highly fragmented along a narrow range on the Andes hillsides. Nonetheless, an exception to this configuration occurs on Tierra del Fuego Island, where N. pumilio forest probably retained or recovered its continuity. Moreover, this forest mass is present at low altitudes (less than 600 m a. s. l.) and is highly influenced by seasonal storm track patterns. In this study the five populations located in Tierra del Fuego were analyzed separately   was found only in GZ14 , and GZ18 BL   Fig. . N. pumiPa Table . AdditioN. pumilio was confirmed by assessing the hierarchical analysis of variance (AMOVA), where the proportion of variance due to both the ‘among- and within-’ group components were used as elements of comparison. Our proposal of eighteen GZs had a greater among-group than within-group component of variance, which is evidence of correct grouping (Table ’ GZs (Table The optimum number of genetic management units (in this case GZs) in ng Table . The phyZs Table  are consN. pumilio was assessed here by neutral molecular markers (SSRs) and was taken into account when delineating N. pumilio GZs in Argentina. This approach has been successfully used to define management units for several species , since microsatellite diversity was correlated with genomic diversity at population level, although not at individual level53. In addition, considering that microsatellite diversity may not reflect adaptive diversity54, when defining Conservation Units (CUs) and Provenance Regions (PRs) it would be appropriate to determine the genetic variation data based on adaptive traits55. Even though Chilean N. pumilio populations were not sampled, the current status of these forests could reflect similar variation to the Argentinean side, since they are probably subject to comparable threats. However, it may be difficult to achieve common management criteria on both sides of the mountain range (Andes Cordillera), since each country has specific regulations.The delineated GZs could facilitate the implementation of specific practices related to conservation programs by determining priority areas based on the genetic diversity of each OGMU. The genetic structure of AR) is commonly chosen as a genetic criterion in order to define priority areas for conservation32. This parameter is independent of sample size56 and suitable for determining historical processes such as bottleneck or admixture56, and inferring putative relictual zones31. Accordingly, GZ2, GZ3, and GZ6 require special attention due to their high genetic diversity , which could be evidence of genotypes adapted to specific environments60. Furthermore, a high LCA value could be indicative of genetic isolation30 and thus help to identify the putative location of species–specific refugia62. Based on the high LCA and/or Pa detected in this study , particular consideration should be given to this area, which is included within GZ9. This demographic event leads to a reduction in genetic diversity, which could threaten the adaptive potential of this population and, in an extreme case, lead to local extinction63. For this reason, strict in situ conservation management is highly recommended for this area, in order to prevent the loss of these genetic variants.In addition, since a relatively recent demographic bottleneck was inferred in the Finally, GZ7 and GZ13 displayed specific genetic composition, based on their genetic structure and admixture patterns. In particular, these zones combine an almost balanced mixture of two surrounding gene pools, and thus, special consideration is suggested for their operational forest management. Local sources must be chosen if reproductive materials are required for active restoration in these GZs, in order to preserve their characteristic genetic structures. In the case of severe loss of the GZ7 and GZ13 local forests , a balanced seed pool from the surrounding GZs is suggested for future tree-planting activities.N. pumilio management plan based on genetic knowledge and considering its entire distribution range (more than 2200 km). Six priority areas  were identified that could lead conservation action, specifically targeting in situ conservation. Furthermore, this study enabled the delineation of 18 N. pumilio GZs in Argentina, thus providing preliminary operational genetic management units that best fit the genetic structure of the target species. Maps showing the distributional range of each GZ in Argentina were drawn up. Reproductive material can thus be transferred within these ad hoc GZs with reduced risk of altering the original genetic structure. This possibility is important in light of the numerous active ecological restoration projects that have been developing in the N. pumilio forests on both sides of the Andes Range in the last decade. In addition, the recommendations suggested here are highly important for regulatory institutions that have to enforce compliance with national Law 26331, on the protection of native forests. Moreover, the 18 GZs constitute a first step towards the definition of both PRs and CUs, thus promoting preservation of the southernmost forest ecosystem of the world.This study presents the first scientific evidence for the definition of a N. pumilio populations (965 adult trees) included in this study  and from 71° 56′ to 66° 37′ in the longitudinal range. A minimum of 20 trees were sampled in each population, with the exception of Cholila and El Chalten sites where 18 and 19 samples were obtained (respectively) due to limited access. In every site, a minimum distance of 50 m between sampled trees was maintained in order to preclude the collection of closely-related individuals. In addition, only N. pumilio phenotypes were considered at the time of population sampling .Of the 35 dy Table , Fig. 1,64. Samples were stored at − 20 °C until PCR amplifications were carried out. Seven polymorphic loci  were analyzed using a specific set of microsatellite markers (nuSSRs) developed for this species by Soliani et al.65. SSR amplification conditions and PCR thermal profiles were set following the recommendations given by the authors65. Amplification reactions were performed with 20 ng of template DNA in a final volume of 11 µl, using 1X Green Buffer and GoTaq DNA Polymerase  for all primers, with the exception of Npum10 that was amplified with Platinum Taq . The M13 protocol66 was applied and PCR products were visualized on 1% agarose gel stained with Sybr Safe (Invitrogen). Individual genotyping was done using a 3130xl Genetic Analyzer sequencer  and the allele assignment with GeneMarker V2.7.4 .Total DNA was extracted from bud tissue following the procedure described by Dumolin et al.Na), the effective number of alleles (Ne), and the expected heterozygosity that considers the null allele effect (HE null), which were obtained with GenAlEx 6.5 software67. Unbiased heterozygosity described by Nei68 was also estimated with Info-Gen software69. The Wilcoxon test was implemented as an a posteriori multiple-comparison analysis, to identify the significant differences among populations. The presence of null alleles at both locus and population levels was tested with MICRO-CHECKER70 and their frequencies were estimated using FreeNA71. The presence of null alleles was considered in the estimation of inbreeding coefficients (FIS), which was calculated with INEST 2.272. Two approaches were tested for obtaining FIS: 1. an Individual Inbreeding Model (IIM) using a Bayesian approach (FIS′), and 2. a Maximum Likelihood approach (FIS”). In both cases, three different models were tested  at population level, and the model with the lowest Deviance Information Criterion (DIC) was considered as best fitting the data. Once the inbreeding model had been selected, the mean inbreeding coefficients (FIS′) were calculated. In addition, a jackknife procedure was performed to estimate the standard error (SE) of the FIS” values, which were then used to assess the significance of this coefficient using the Z-test based on the normal distribution theory.A set of genetic parameters was first obtained in order to characterize the populations under study Table . These p73. This approach computes the distribution of the expected heterozygosity from the observed number of alleles at population level, assuming the mutation-drift equilibrium model. A significant heterozygosity excess or deficiency is confirmed by performing a Wilcoxon sign-rank test for every mutation model . In this study, a past population bottleneck was inferred when heterozygosity excess was significant (p < 0.05) in at least two of the above-mentioned mutational models.In order to identify populations experiencing a relatively recent reduction in effective population size, a demographic bottleneck test was carried out using BOTTLENECK softwareFST74 and G′ST75 indexes. FST was then recalculated by implementing the ‘exclusion of null alleles’ (ENA) method in order to avoid a possible bias introduced by the presence of null alleles71. Confidence intervals (95% level) were obtained through a bootstrap re-sampling procedure. In addition, to evaluate the occurrence of an isolation-by-distance (IBD) pattern, a correlation between the genetic and geographic distances between populations was assessed using two approaches: a. The Mantel test, performed with Info-Gen and using the correlation between FST estimates and the logarithm of the geographic distances between population pairs; b. The Spatial Genetic Structure (SGC) test by GenAlEx 6.4, with which spatial autocorrelation coefficients  were obtained for ten distance classes (each one covering 200 km). In both approaches, 1000 permutations were considered when calculating statistical significance. In the SGC test, the upper and lower error limits for the 95% confidence interval were determined by carrying out 10,000 bootstrap samples.Genetic differentiation among populations was determined by estimating both 33, see Fig. 77. This analysis showed weak grouping, since the adjusted probability of the inferred number of clusters was 0.58632. This reinforced the decision of considering alternative division criteria before performing the final clustering.Due to the large latitudinal span of the area under study (more than 2200 km long) and the diverse evolutionary history of the region, multiple division criteria were considered  and Cholila, and 2. ‘South’ including the remaining 23 populations that exhibited southern cpDNA haplotypes and are located between Huemules and Bahía Lapataia (54° 50′S). The genetic structure of N. pumilio populations from each north and south group was then determined by a spatial clustering method using BAPS software. This approach has the advantage of considering the geographic locations of pre-assigned groups of individuals when defining the best number of clusters (K)77. In addition to this analysis, an admixture model within each inferred K was tested with STRUCTURE 2.2 software79 by running 500,000 Markov chain Monte Carlo (MCMC) steps, a burn-in of 100,000 iterations and five replicates for each inferred K in the entire analysis. Complementarily, UPGMA (Unweighted Pair Group Method using Arithmetic averages) dendrograms based on Nei’s distance (averaged over loci) were constructed to explore the relationships among the identified clusters.A phylogeographic break around 42°S latitude was previously reported for some species of 32 and Thomas et al.80. In this case, individual admixture coefficients for each inferred cluster (obtained by STRUCTURE) larger than 0.7 were considered. Each cell was assigned with the mean membership value of the individuals, enclosed by a circular neighborhood of 20 min diameter constructed around its center to graphically represent the inferred clusters. Raster package 2.14 was used to generate these maps in R software environment (R Core Team) and they were edited in ArcGIS 9.3 .A grid-based spatial analysis was carried out with a resolution of 2 min to visualize the geographical distribution and admixture pattern of inferred clusters. Separate raster maps were produced, considering a circular neighborhood cell of 20 min diameter around every sampled location, following van Zonneveld et al.Delineation of GZs was performed based on the previously-mentioned analyses and summarized in Fig. 81, in order to evaluate the number of clusters that best grouped the studied populations. The optimum number of genetic zones was confirmed based on the significant differences among and within the obtained clusters, as proposed by Pastorino and Gallo20; i.e., genetic variance should be maximum between populations of different groups and minimum within the groups.A hierarchical analysis of molecular variance (AMOVA) was performed using Arlequin 3.5Pa), the expected heterozygosity considering the null allele effect (HE null), and locally common alleles (LCA). The LCA average for each GZ was calculated considering alleles with frequencies higher than 5% and, at the same time, an occurrence probability of less than 25% of the defined GZs. In addition, genetic diversity was estimated by calculating allelic richness (AR) after rarefaction to a common sample size82 using FSTAT software83.To obtain valuable information for the definition of management plans, some genetic parameters for each defined GZ were calculated with GenAlEx and Info-Gen: private alleles (Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


As Novel psychoactive substances (NPS) are conceived to mimic the effects of common illicit drugs, they represent a serious public health challenge due to the spike in intoxications and fatalities that have been linked to their use. This study aims to provide epidemiological data on NPS use in the USA, determining lifetime prevalence of use and defining demographic, socioeconomic, drug use patterns and mental health correlates.This study uses secondary data from the US National Survey on Drug Use and Health (NSDUH), which is a large cross-sectional population-based survey carried out annually in the USA. We analysed data from 2007–14  using bivariate descriptive analysis and binary logistic regression to calculate prevalence and determine factors underlying NPS consumption. Adjusted odds ratios (OR) with 95% CI’s were calculated for a set of selected independent variables.Our analysis NSDUH from 2007–14 highlights an increase in NPS use among adults, especially among white young men aged 18 to 25. Although the level of education of NPS users was relatively higher as compared to non-users, NPS users seemed to have a less wealthy situation. However, socioeconomic vulnerability appeared to be less important than mental health issues as a correlate to NPS use. NPS users seem to have followed a pattern of polysubstance use throughout their life, which involves both traditional illicit drugs and classic synthetic drugs. As NPS use seemed to be more prevalent among people having mental health issues, the rise in their use may have a negative impact on population mental health outcomes.Further comparative research on trends in NPS use and potential public health responses would be instrumental for developing appropriate health interventions, including drug checking, education for users and training for healthcare professionals working both within emergency wards and in/outpatient addiction and mental health services. Over the last few decades, the emergence of Novel Psychoactive Substance (NPS), sometimes also called “legal highs”, “research chemicals” or “designer drugs”, has become a topic of interest for researchers, policy officers, healthcare providers and enforcement agencies in high-income countries –9. NPS aN = 307,935). This eight-year timeframe was chosen due to notable changes made to the survey questionnaire and data collection procedures in 2015, affecting comparability over time [via face-to-face interviews using computer-assisted interviewing (CAI) and audio computer-assisted self-interviewing (ACASI) to increase respondents’ cooperation and willingness to report honestly about topics such as illicit drug use behaviour and mental health issues. Questions pertaining to the use of regulated substances are self-administered through ACASI [This study analyses secondary data from the NSDUH. It is therefore no primary recruitment of participants since all data has already been collected nor it is possible to obtain consent from participants included in this study. Data for adults aged 18 and over were included from the 2007–14 cohorts of the NSDUH, an annual, cross-sectional population-based survey of civilian, non-institutionalised individuals (ver time . The samgh ACASI .th grade/Freshman or higher). Marital status was coded as either Single–including widowed, divorced and separated–or Married. Metropolitan size was coded as Large, Small or Non-metropolitan based on 2010 census data and 2009 Core Based Statistical Area classifications provided by the Office of Management and Budget (OMB) [We categorized individuals from the NSDUH according to the following age groups: 18–25, 26–34, 35–49 and 50 years or older. Sex was coded as either Male or Female. Respondent ethnic background was coded as White, African American, Hispanic and Other (including Native American and Asian or Pacific Islander). Education was coded along the following categories: Elementary (7th grade or less), Secondary (8th to 12th grade), and Post-Secondary (13et (OMB) .Employment status was categorized as Full-time, Part-time, Not employed (including unemployed and not in labour force). Household annual income was categorised as: less than $20,000; $20,000 - $49,999; $50,000 - $74,999; $75,000 or more. A dichotomous variable was created to indicate whether a respondent received any form of government assistance . The respondent’s primary health insurance was coded as Private, Medicare , Medicaid , Tricare & Veterans Administration (VA) (for military veterans), other, or Uninsured. Additionally, based on the four indicators described above, an aggregate index of socioeconomic vulnerability was created by summing the number of vulnerability attributes observed . The index values range from 0 (which indicates no vulnerability attributes) to high (when at least 3 vulnerability attributes were observed).We considered lifetime use of 64 NPS as our outcome variable. These substances were spontaneously mentioned by respondents through open-ended questions about the recreational use of (1) prescription drugs , (2) hallucinogens , (3) inhalants, (4) non-prescription cough or cold medicines and (5) special drugs such as injectable stimulants. For the purpose of this study, we defined as NPS according to two criteria: 1) their international legal status (substances not classified under Schedules I and II of the 1971 UN Convention on Psychotropic Substances); 2) their recreational use among general population, defined by the questions about their consumption for the experience or feeling they cause (“for kicks or to get high”). A few exceptions to these rules were made with controlled substances whose recreational use has remained limited until recently , MDA, PMA) .We listed 63 reported substances and classified them into four NPS categories according to their pharmacological effects: (1) hallucinogens, (2) stimulants, (3) depressants and (4) synthetic cannabinoids. Among the 64 substances reviewed, we identified 29 phenethylamines , 17 tryptamines, 5 synthetic cathinones (including 1 generic category encompassing three different street names), 3 synthetic cannabinoids (including 1 generic category compassing nine different street names), 2 ergolines, 2 dissociatives, 2 benzodiazepines, 1 aminoindane, 1 piperazine, 1 inhalant and 1 synthetic opioid . We subsIn order to better characterise the drug use patterns of NPS users, two aggregate dichotomous variables were created. Similar to the construction of the lifetime NPS use variable, we created an aggregate indicator for lifetime use of illicit drugs based on the use of at least one of the four traditional illicit drugs included in the NSDUH. This aggregate indicator was constructed based on the data collected through the NSDUH core questions on lifetime use of cocaine, crack, heroin and cannabis. Additionally, an aggregate indicator for lifetime use of “classic synthetic drugs” was also constructed based on the data from NSDUH core questions on lifetime use of LSD, ecstasy and PCP, as well as the use of so-called (2) “special drugs” such as methamphetamine and GHB, as defined by NSDUH . For thePast-year prevalence of four mental health indicators were included in the study. First, we used the Serious Psychological Distress variable (SPD), which is based on the Psychological Distress Scale (K6) that gathers information regarding how frequently individuals experience symptoms of psychological distress over a time period (e.g. over the past month or over the worst month of the past year that was not the past 30 days), with values ranging from 0 to 24. Symptoms of distress include feeling nervous, feeling hopeless, feeling restless or fidgety, feeling so sad or depressed that nothing could cheer one up, feeling that everything was an effort, and feeling down on oneself, no good, or worthless. For each of these six items, responses of "all of the time" were coded 4, "most of the time" were coded 3, "some of the time" were coded 2, "a little of the time" were coded 1, and "none of the time" and all other responses were coded 0. These assigned values were summed across the six items to calculate a past month K6 total score and a K6 score for the worst month of the past year other than the past 30 days. Respondents were classified as having had a past year SPD if their K6 score was 13 or higher . Conversely, they are classified as not having had a past year SPD if their K6 score was lower than 13 .Second, we used the past-year Major Depressive Episode (MDE) indicator. Respondents were defined as having MDE in the past year if they had a lifetime MDE and a period of time in the past 12 months when they felt depressed or lost interest or pleasure in daily activities for 2 weeks or longer, while also having some of the other symptoms for lifetime MDE. Respondents were defined as having had a lifetime MDE if they had five or more of nine symptoms for MDE in the same 2-week period in their lifetime, in which at least one of the symptoms was a depressed mood or loss of interest or pleasure in daily activities. Symptoms for MDE include: (1) depressed mood most of the day; 2) markedly diminished interest or pleasure in all or almost all activities most of the day; 3) changes in appetite and weight; 4) insomnia or hypersomnia; 5) psychomotor agitation or retardation; 6) fatigue or loss of energy; 7) feelings of worthlessness; 8) diminished ability to think or concentrate or indecisiveness; 9) recurrent thoughts of death or recurrent suicide idea. Respondents with no lifetime MDE or respondents with lifetime MDE but no period of depression lasting 2 weeks or longer while having other symptoms were defined as not having past year MDE. It should be noted at this point that the NSDUH makes no exclusions for MDE caused by medical illness, bereavement, or substance use disorders .Third, we created an indicator of past-year access to mental health treatment that encompasses access to in/outpatient mental health treatment and use of prescription medicine for a mental health condition . FinallySince 1999, the NSDUH addresses item nonresponse using an imputation method known as predictive mean neighbourhood (PMN), which is applied in a stepwise fashion: (1) response propensity adjustment; (2) prediction modelling; and (3) hot-deck imputation . Data imp<0.05; yet it appeared that none of the factors had a strong correlation with the outcome variable. As a result, we performed a binary logistic regression for NPS use and a set of independent variables selected upon our previous knowledge of the topic. Adjusted odds ratios (OR) with 95% CI’s were calculated for the selection of the independent variables included in the model . As the available epidemiological evidence on the use of NPS is still scarce, one of our objectives was to provide relevant population-based data. Hence, we decided to use adjusted odds ratios (OR) as they are commonly used in epidemiology to compare the relative odds of the occurrence of a particular outcome (e.g. NPS use), given exposure to a series of variables of interest . Although, the 95% confidence interval (CI), used to estimate the precision of the OR, may not be the most adequate measure of accuracy for the purpose of this study.All statistical analyses were performed in Stata 14.2. NSDUH datasets from individual years were combined into a single file for analysis to allow for year to year comparisons. In order to estimate the annual average of the number of individuals who engaged in a particular behaviour based upon pooled data from 2007–14, adjusted analysis weights were created by dividing annual weights by eight (the number of cohorts under study) according to the NSDUH Public Use File Codebook . Thus, aConsumption of NPS showed an 167% increase between 2007–14. In 2007, the estimate for lifetime NPS use among adults was 0.09% , while 0.24%  of adults had used an NPS seven years later .The increase in NPS use was notably led by young people–particularly by those between the ages of 18 and 25 –whose lifetime prevalence of NPS use exhibited a fourfold increase between 2007–14. The exception to this upward trend was the pattern followed by people between aged 50 or older, whose prevalence of NPS use remained stable at around 0.1% during the same period .Regarding the evolution over time by NPS category, it is worth to note that the use of synthetic cannabinoids rose steadily between 2007–2014: it accounted for 0.8% of reports in 2008 (when it was first mentioned by respondents), reaching up to 17.1% in 2014. On the other hand, the use of hallucinogens and stimulants showed a downward trend during the same period (-11% and -8% of reports respectively). Meanwhile, the use of depressants remained stable at around 1% of reports .Measures of lifetime NPS use were obtained for four NPS categories. The differences in the proportions of these categories were highly significant, with hallucinogens being the most frequently reported by NPS users (73.8%), followed by stimulants (13.9%) and synthetic cannabinoids (11.7%). The use of depressants (0.6%) appeared to be less widespread among our study population . The oveAccording to our results , NPS useThe large majority of NPS users were active workers: 49.94% of them work on a full-time basis (95% CI = 44.37%-55.51%) and 25.29% had a part-time job (95% CI = 19.94%-31.50%). As NPS use still was a rare phenomenon during the period under study, comparisons with non-users of NPS and general population appear to be equivalent.The proportion of people having a household annual income lower than $20,000 was slightly higher among NPS users  than among non-users . Similarly, the proportions of people without health insurance  and receiving government assistance  were relatively higher among NPS users as compared to non-users . Consequently, the proportion of people showing no factors of socioeconomic vulnerability as defined by our vulnerability index (cf. 2.3) was lower among NPS users  than among non-users . Finally, the use of NPS was more common among young adults under 35 years old, particularly among those aged 18 to 25 that represent about 4 in 10 NPS users. Together, young adults aged 18 to 34 represented 63.76% of NPS users, while they only represented 30.45% of non-users . In addiNPS users with the highest level of education were twice more likely to use an NPS (adjusted OR = 2.46). Conversely, the use of NPS was significantly less likely among people aged between 35–49  and among African Americans  .Different trends in NPS use observed among age groups. The use of hallucinogens was predominantly reported by people aged 34 or younger, especially by young adults under 25 . Meanwhile, the use of stimulants seemed to be more common among people aged 50 or older (54.7% of them reported use of NPS stimulants). Interestingly, as illustrated by As shown in Additionally, 92.43% (95% CI = 89.56%-94.56%) of NPS users had also already used classic synthetic drugs, particularly LSD  and ecstasy . The use of PCP , GHB  and methamphetamine  appeared to be less common among NPS users . Overallp < 0.000) and those that had used traditional illicit drugs were 7.67 times more likely to do so   .NPS use seemed to be more frequent among adults who had experienced mental health problems. While the overall estimate of lifetime NPS use for adults was 0.17%, it reached 0.42% (95% CI = 0.33%-0.52%) among adults who experienced serious psychological distress (SPD) in the last year and 0.45% (95% CI = 0.34%-0.58%) among those who had a major depressive episode (MDE). Moreover, the estimate for lifetime NPS use was 0.32% (95% CI = 0.25%-0.41%) for adults who received mental health treatment during the last year, while it was 0.51% (95% CI = 0.39%-0.65%) among those that had perceived an unmet mental health need .As shown in According to our findings, the lifetime prevalence of NPS use increased between 2007–14, especially among young adults aged 18 to 25. Although the level of education of NPS users was relatively higher as compared to non-users , NPS users seemed to have a less wealthy situation. NPS users seemed to have followed a pattern of polysubstance use throughout their life, particularly of cannabis, cocaine and classic synthetic drugs. This is consistent with findings from prior international studies that have already pointed out patterns of polysubstance use among NPS users . As mostThe profile of NPS users that may be addressed based on the results of our study  seem to be concordant with results from recent studies conducted in Europe among NPS users that identify young active workers using NPS to cope with their daily life challenges and dealing with health issues as one of the various profiles of NPS users –54.It is noteworthy to emphasise a few limitations of this study. First, the overall objective of the NSDUH is to provide data on the level and patterns of alcohol, tobacco and illegal substance use and misuse, while the present study particularly aimed to look at the use of NPS based on the data obtained through open-ended questions. In addition, commonly used data collection strategies are known to be inefficient for rare events data such as NPS use, while statistical procedures such as logistic regression, can considerably underestimate the probability of rare events . Hence, Second, the use of self-reported substance use as an outcome variable may have led to significant levels of mis/underreporting due to the diversity of substances currently available in the drug market , the variety of nomenclatures associated with them, and the lack of awareness among respondents of the actual chemical composition of the substances they used. Furthermore, the design of the NSDUH as a household survey does not permit sampling of homeless or institutionalized persons, who show specific patterns of problematic drug use , 57. FinThe increase in NPS consumption observed over the last decade has raised public health concern about the risks related to the consumption of new substances whose actual origin and composition remained widely unknown. The emergence of NPS is particularly worrying given the scarcity of evidence around the pharmacology and potential toxic effects of new psychoactive substances regularly appearing in the international drug market. Although the coexistence of both problematic drug use and mental health issues is a well-known phenomenon within the scientific community, in the USA this new trend in drug use is of particular interest considering that the proportion of reported unmet mental health need seems to be higher among NPS users as compared to non-users. Further comparative research on trends in NPS use and potential policy public health responses would be instrumental for developing appropriate health interventions, including drug checking, education for users and training for healthcare professionals working both within emergency wards and in/outpatient addiction and mental health services.S1 Table(DOCX)Click here for additional data file.