Agave. Amongst them, A. angustifolia is the most popular given its agricultural extension. A. angustifolia is a wild species extensively distributed from North to Central America, and previous studies show that it is highly related to the tequila agave A. tequilana. In different regions of Mexico, A. angustifolia is cultivated under different types and levels of management, and although traditional producers identify several landraces, for the non-trained eye there are no perceivable differences. After interviews with producers from different localities in Jalisco, Mexico, we sampled A. angustifolia plants classified as different landraces, measured several morphological traits, and characterized their genetic differentiation and diversity at the genome-wide level. We included additional samples identified as A. tequilana and A. rhodacantha to evaluate their relationship with A. angustifolia. In contrast with previous studies, our pool of ca 20K high quality unlinked SNPs provided more information and helped us to distinguish different genetic groups that are congruent with the ethnobotanical landraces. We found no evidence to genetically delimitate A. tequilana, A. rhodacantha and A. angustifolia. Our large genome level dataset allows a better understanding of the genetic identity of important A. angustifolia traditional and autochthonous landraces.Traditional agave spirits such as mezcal or tequila are produced all over Mexico using different species of  Agave for spirits production is recent in human history. Although it has been suggested that Agave plants have been used as a source of food or fiber production for at least 9000 years [Agave did not begin until the 16th century and the cultivation and production of spirits increased with the popularity of tequila as recently as the early 19th century [Agave with spirit production purposes, especially for some landraces such as the tequila plant. This, together with the long life-cycle particular of agaves, explains the incipient domestication status of cultivars and landraces.The use of 00 years ,2, the d century ,5,6,7,8.Agave, tequila is the most emblematic. Made from the “blue agave” plant Agave tequilana var. “Azul”, tequila has been produced traditionally since the 18th century in the state of Jalisco . As it happened with tequila, mezcal has also gained worldwide recognition and explosive demand. Over the period between 2011 and 2019, mezcal production in Mexico increased over 700% [Mexico is the center of the diversity and domestication of Agave , where 5Agave ,23. Howe complex ,25. Prevue agave ,18,26. Eoduction . Anotherver 700% , with thver 700% . Under tA. angustifolia complex have been studied using traditional genetic markers such as AFLPs and microsatellites [Agave [A. angustifolia complex in Jalisco. We visited 12 mezcaleras both in the North Coast and in Southern Jalisco and interviewed with local producers. We sampled several individual plants that were referred to by them as different landraces or semi-wild landraces . We also sampled A. angustifolia plants in the wild, as well as additional A. tequilana and A. rhodacantha accessions, to evaluate their genetic identity and characterize their genetic diversity in relation to geography and ethnobotanical characterization. Finally, we conducted several morphological measurements on a subset of our cultivated sampled plants that were classified by producers as belonging to the widest recognized landraces. This study contributes with the largest available dataset at the genomic level for local Jalisco traditional landraces. Our pool of SNPs allowed us to better understand the genetics underneath the cultivation, propagation and domestication processes of species, varieties, and landraces within the A. angustifolia complex in the region.Although some species and landraces included in the tellites ,24,25,26tellites , and it s [Agave . In thisA. tequilana var. “Azul” allowed us to generate a matrix of 67,175 high-quality SNPs, with a mean individual SNP coverage of 0.91%. Linked SNPs within a window size of 50, a step of 10 and square-r threshold of 0.1 were pruned, leaving a final matrix of 19,983 SNPs after filtering, suggesting about 70% of the original SNP dataset was linked. According to this dataset, the A. angustifolia Jalisco complex studied with our samples showed low genetic variation, with a mean expected heterozygosity value (eH) of 0.1311 ± 0.0110 and mean nucleotide diversity (π) of 0.2622 ± 0.0220.In total, we collected 87 samples in the state of Jalisco, Mexico between 2020 and 2021 . With ouSTF mean value (0.3747 ± 0.0740) showed moderate-to-high genetic divergence across individuals. To further explore the population structure, we carried out an Identity by State (IBS) analysis to compare pairwise genetic distances. We did not find a full congruence of the clusters generated using genetic distances with the landraces. To verify, we conducted an Identity by Descent (IBD) test. Without exception, all pairwise IBD values suggested each sample is genetically related to each other. Therefore, we omitted genetic distances in the following procedures.The pairwise We carried out a Principal Component Analysis (PCA) using our filtered SNP dataset. The first three components of the PCA explained 13.9321%, 11.1120% and 10.4036% of the variance, respectively. The Euclidean distances of the eigenvectors from the three components were used to build a UPGMA genetic dendrogram . Four maA. tequilana var. “Chato”. Cluster 4b is the most complex of all clusters. It includes several different landraces as well as plants from the wild. The most divergent sample within 4b is TET5, classified as “Lineño” by producers in Tetiapán . Two subgroups in addition to TET5 can be found within 4b. The first includes all samples from different landraces in the North Coast, all collected in Cabo Corrientes: “Cenizo” , “Amarillo” (PDSA), and “Chico Aguiar” . The other subgroup includes all samples of plants from Southern Jalisco: samples collected in the wild from Apango (coded as AP) and Sayula (coded as SAY), several plants classified as “Cimarron” collected in Zapotitlán de Vadillo , Tolimán (TOL4), Tetiapán (TET2); “Negro Cimarrón” from Zapotitlán de Vadillo (NCZ) and Tolimán (NCMG1), “Cimarrón Verde” from Tolimán (CVMG2), “Azul Telcruz” from Zapotitlán de Vadillo (ATLA9) and Tolimán (SOMG1), “Lineño” from Tetiapán (TET6), “Ixtero verde” from Zapotitlán de Vadillo (IVLA3); as well as A. tequilana var. “Mano Larga” (C1ML1) and A. tequilana var. “Azul” (CIAT1-2).The fourth cluster is the largest, comprising a cohesive group with highly related samples, mostly coming from plants growing in the wild. This group comprises a more genetically diverse set of samples from different regions. It is split in two major subgroups: 4a and 4b with excess of homozygotes and the highest values of Pi . ClusterK values from two to eight and ran ten repetitions with a random seed each. We observed that K = 5 had the lowest cross-validation error rate from the analysis. Then, we looked for the most representative repetitions with CLUMPAK. We selected the fifth repetition from K = 2 (7/10), the first repetition from K = 4 (8/10) and the eighth repetition from K = 5 (10/10) , we noted A. tequilana and A. rhodacantha landraces such as “Chato”, “Azul” and “Chico Aguiar” are fully represented.The arrangement of our samples in the genetic clusters described above was also congruent with the results of the analyses of the genetic structure with ADMIXTURE. For these analyses, we selected /10) see . When K Agave landraces [We gathered morphological data for 20 genotypes in our dataset , focusing on several variables that have been useful to study traditional andraces . In totaThe measurements were used to build a heatmap a, which Using our morphological measurements, we built a UPGMA dendrogram based on Euclidean distances. This dendrogram showed groups that are highly congruent with landrace determination as well as geography a. Three p-value = 0.001) and the derived dendrograms a cophenetic and Baker matrix correlation of 0.54 and 0.7, respectively , showing high equivalence among them.The dendrogram built with morphological data was compared both visually and numerically (cophenetic and Baker correlation matrix indexes) with a genetic tree built with a reduced SNPs dataset that only includes the samples for which we had morphological measurements. This last dendrogram of genetic distances shows highly cohesive and supported grouping patterns that follow landrace classification. However, there is no full congruence amongst morphological and genetic dendrograms, as the congruence is more evident only in some groups b. Genetipers. com). Even though producers identify several landraces, only few are commonly known and less ambiguously identified by most of them in each region, which might indicate the generational time elapsed since the start of management and the strength of their genetic identity. That is the case of landraces “Amarillo”, “Verde”, “Cenizo” and “Chico Aguiar” in the North Coast, as well as “Ixtero Amarillo”, “Ixtero Verde” and “Lineño” in the South. In contrast, other plants grown in these agroforest systems are classified using terms such as "Cimarrón”, “Sierreño” or “Barranqueño”. These terms refer to plants that farmers recently found in the wild and identified as good candidates to bring to their cultivars. This is the mechanism by which landraces probably start and then producers keep reproducing them asexually for generations. For example, the terms “Negro Cimarrón” or “Cimarrón Verde” indicate plants that have recently been brought from the wild or are under incipient management. Plants under these different stages of management suggested by their ethnobotanical denomination might show different levels of genetic diversity and differentiation [As it happened with the history of tequila production in the region of west-central Mexico, the specific mezcaleras visited for this study began to be managed at least during the second half of the 18th century  in comparison with previous studies using traditional markers such as AFLPs or microsatellites [A tequilana, A. angustifolia and A. rhodacantha, but do not show a clear differentiation of these three species in separate supported clusters. In contrast, our samples of A. tequilana and A. rhodacantha or A. aff. rhodacantha are mixed and intercalated amongst a diverse arrangement of A. angustifolia wild and landraces samples, suggesting the extensive gene flow and poor genetic limits existing amongst entities within the complex. However, a more conclusive assessment of the identity of these taxonomic units and landraces requires the inclusion of more specimens from different localities in the study, as well as morphological data for both vegetative as well as reproductive structures.The oduction . In thistellites ,32. Our A. angustifolia complex coming from AFLP studies showed the genetic differentiation but very close relationship of A. angustifolia and A. tequilana, even suggesting that A. tequilana is a type of A. angustifolia [A. tequilana as a cohesive group closely related to A. angustifolia var. “Espadin” [A. angustifolia growing on the semi-arid slopes in Cocula and Tecolotlán in Central Jalisco [Previous information on the genetics within the stifolia . AdditioEspadin” . These r Jalisco ,8.A. tequilana and the suggested landraces have been studied in more depth than A. angustifolia. The study of the genetics of A. tequilana with traditional genome-wide genetic techniques such as AFLPs and RAPDs were inconclusive, showing either low or significant diversity depending on the technique used [A. angustifolia complex in Jalisco, finding that A. tequilana var “Azul” samples were more closely related to the A. angustifolia populations from southern Jalisco. In addition, they found that the A. tequilana var “Sigüin” and A. tequilana var “Chato” as well as the landrace “Ixtero Amarillo” are closely related.Given its importance in tequila production, que used ,32. Thesque used , or evenque used . By usinque used  studied A. tequilana var. “Chato” , and two accessions of A. tequilana var. “Azul” , as well as one accession of A. tequilana var. “Mano Larga” (C1ML-1). Interestingly, and similarly to Trejo et al. [A. angustifolia backgrounds. We also found more genetic variation in these A. tequilana landraces than previously thought, which deserves more in-depth studies. Our results indicate that the A. tequilana var. “Chato” in the cluster 4a , a region located to the north of Guadalajara city, geographically close to Tequila, the locality of origin of the blue agave. On the other hand, our samples of A. tequilana var. “Azul” and A. tequilana var. “Mano Larga” both group in a totally different cluster, 4b, which is the largest in our study and includes several landraces and wild plants collected in the south of Jalisco, suggesting that these two other A. tequilana landraces might have their origins in that region.For our study, we included in our analyses two accessions corresponding to the o et al. , our resuster 4a  emerged Agave. In most localities, particularly where spirits are distilled traditionally, producers still recur to wild plants and follow locally developed non-industrialized methods. In other regions where the production of spirits has deeper heritage and history, families of farm holders and producers inherit a tradition of cultivating and propagating plants that were initially brought from the wild generations ago. It has been suggested that landraces such as “Ixtero Verde”, “Ixtero Amarillo”, “Cenizo” or “Lineño” have been cultivated in the region from at least 100–150 years [Agave spirits are produced all over Mexico, applying both industrialized or semi-industrialized as well as traditional and artisanal methods using different species of 50 years .Agave spirit producing crops provide particularities to their domestication processes and establishment of landraces. The long life- cycles of Agave plants (around 6 to 10 years or more), and the fact that reproductive structures that allow sexual crossing are cut before maturity during their cultivation, make breeding practices practically inexistent. Plants recognized as landraces might come from one or a few genetic lines of plants found originally in the wild, and then propagated over and over asexually through clonal rhizome plantlets or bulbils. Thus, we expect the genetic variation within landraces to be significantly reduced. We could also expect that morphological characteristics are highly consistent; however, the recognition and naming of landraces by producers involves a social process of acceptance of the group of morphological traits, particularly if it occurs amongst different farm holders over larger geographic areas. For example, although most landraces are cultivated by single producers, others such as “Lineño” and “Ixtero Amarillo” are more widely recognized and distributed, suggesting a longer history of propagation and cultivation [Some inherent biological characteristics of tivation .A. angustifolia complex, the morphological characteristics of several landraces have been studied extensively in Jalisco by Vargas-Ponce and collaborators. These authors report the presence of at least 24 traditional landraces cultivated in the state, most of which are grown in Southern Jalisco. In contrast, Central Jalisco is currently dominated by monovarietal plantations of A. tequilana var “Azul” for tequila production, and traditional landraces have become very scarce [A. angustifolia landraces in Guerrero, Mexico using a similar set of variables, and they were also able to separate the individuals belonging to populations of the “Sacatoro” landrace from the population of the “Espadín” landrace included in their study. With this set of morphological variables, we were also able to confirm that the studied landraces already show morphological differentiation and cohesiveness at this organismic level, showing the adequacy and sufficiency of the morphological variables selected to study the differentiation of landraces in the A. angustifolia complex in Jalisco, as well as in other regions of Mexico.In the case of the y scarce . Our stuy scarce  study A.Although with a limited sampling, our analyses of morphological data clearly differentiated landraces that form strongly supported clusters: “Cenizo”, and “Chico Aguiar” from the North Coast and “Ixtero Amarillo” from the South b. The plA. angustifolia complex assigned to several autochthonous landraces under different levels of acceptance and width of use in Jalisco. We also included a significant number of wild plants from the same geographical region, which allowed us to have a better picture of the domestication process in the wild genetic context. With the genomic information obtained in this study, we were able to fine discriminate among producer’s varieties, showing that several landraces have a genomic basis. The use, preservation and promotion of these traditional Agave landraces has been proposed as an alternative to reduce the impact of the extensive monovarietal cultivation of A. tequilana var. “Azul” [Our sampling design allowed for the collection and comparison of wild plants and plants growing under different levels of domestication and management  at the genomic level. We generated a set of approximately 20K high-quality SNPs by RAD sequencing. The restriction site associated (RAD) DNA sequencing is one of the most common methods to genotype by sequence representative loci from all the genomes in plant populations . With ou. “Azul” .Agave species and landraces have been used in the Jalisco and surrounding regions for at least 2500 years, and that they have been very important in both daily and ceremonial life [Agave spp., and refers to the first use of this specific landrace [pers. com).Archaeological evidence shows that ial life ,37. For landrace , with thlandrace . In thisBased on genomic data, our results show a clear differentiation in the landraces that are best recognized by farmers: two in Southern Jalisco (“Ixtero Amarillo” and “Lineño”) and one in the North Coast (“Amarillo”), that conform separate supported clusters , even when the plants were sampled from different parcels or farms. These groups, particularly “Amarillo”, show small Ho values , indicatpers. com). In contrast with the scenario found in Southern Jalisco, there are no historical or anthropological studies on the management of Agave in the Cabo Corrientes municipality. However, producers share similar traditions with the ones in Southern Jalisco, such as the Filipino distillation method, which incorporates stills made with hollow trunks. In both regions, producers refer to their spirit factories as “taverns” and the fermented beverage that they distillate as “tuba”. Thus, the clear genomic differentiation of “Amarillo” and common cultural traits related to the elaboration of spirits between the North Coast and Southern Jalisco suggest a similar human selection process in both regions and are congruent with the hypothesis of Bruman [The “Amarillo” landrace, the most clearly differentiated landrace in our study both genetically and morphologically, sustains 95% of the Raicilla spirit production on the North Coast . This lA. angustifolia var. pacifica in Sonora, the north of Mexico [Finally, samples that came from landraces used with less frequency by producers are all grouped within the large Cluster 4 , left, wf Mexico . This isA. angustifolia complex, together with a more extensive study of landraces with an increased sampling, will allow the detection of loci of interest related to the desired valuable traits, as well as a correlation to regional abiotic conditions.Selection criteria for spirit production vary between producers. Some valuable traits are linked to the production of larger, heavier heads, high sugar content, resistance to pests, diseases and foragers, reproductive precociousness, flavor, or meeting commercial criteria . The genA. angustifolia autochthonous landraces using a GBS approach. This geographic area is highly relevant because it holds the greatest diversity of landraces in the A. angustifolia complex; it is also the putative center of origin of agave distillation in general, and the domestication of the blue agave tequila plant in particular. Our approach, using GBS sequencing and SNPs calling, gave us access to a significantly larger number of loci to analyze in comparison with previous studies using traditional markers such as AFLPs or microsatellites. Our data show that the more extensively used and widely recognized traditional landraces such as “Ixtero”, “Lineño” or “Amarillo” have an important degree of differentiation, both at the genetic and morphological level. However, the differentiation is not absolute and there is a very close relationship with wild genotypes. This might be the result of the extremely recent management and domestication of landraces, together with the important use of clonal propagation. We document the genetic basis of the domestication process in the area, where farm holders recur to wild plants to select and propagate new local varieties that reduce their genetic diversity after several generations of clonal propagation. This method promotes the acquisition of locally adapted germplasm and the diversification of available landraces.In this study, we report for the first time a genome-wide characterization of Jalisco spirit producing The information we provide enables a better characterization of traditional landraces in the state of Jalisco, Mexico. We hope that it helps in technologizing their use and management and provides tools to foment and protect traditional landraces and farming systems to preserve the agrobiodiversity they hold.A. tequilana var. “Azul”. When sampling cultivated plants, producers were interviewed to document the landraces they use and how they recognize them. With their help, we selected several mature plants per landrace and took morphometric measurements. Our pool of samples includes 14 landraces from both A. angustifolia and A. rhodacantha .In total, we collected 87 samples in the state of Jalisco, Mexico during 2020 and 2021 . We visidacantha . AdditioA. angustifolia plants growing in the wild. In this case, plants sampled were located far enough from farmsteads, far from residential or agricultural properties, and evidently growing under isolation and not under cultivation. When wild plants were isolated, we collected only tissue samples for DNA extraction. When wild plants were found in colonies or populations, ten individuals separated by more than 10m were sampled and an herbarium voucher specimen was prepared . The morphological variables measured have previously shown to be useful in the study and characterization of traditional agave landraces : plant l® Kit). Genomic DNA extracted was checked for degradation using a 1% agarose gel electrophoresis, and the quality was determined using NanoDrop 8000 ). Samples were standardized and library preparation was performed at the Genomics Services Laboratory in Langebio, Mexico. Samples were double digested using the restriction enzymes BglII y DdeI and sequenced using the NovaSeq Illumina platform for 5 million single end 100bp reads per sample (1 × 100).Tissue samples of plants collected in the field were either immediately frozen on liquid N or maintained at 4 °C for 24 to 36 h and then transferred to a −70 °C storage. Approximately 300 mg of each sample was grounded using liquid nitrogen and high-molecular-weight DNA was extracted  (in prep) and was used as a reference to align sequenced reads using the default algorithm of the Burrows–Wheeler Aligner v0.7.15 , and th were calculated using 1000 bootstrap repetitions, p-value STFcorrection and kernel smoothing .Population genetics statistics were computed using the populations program in STACKS v2.3e  with thehttp://clumpak.tau.ac.il/; accessed on 14 May 2022 [He), observed Heterocigosity (Ho) and inbreeding coeffient (Fis) were calculated with the R packages hierfstat [To select representative unlinked SNPs, the original dataset was pruned by linkage disequilibrium (LD) using PLINK v1.9  with staMay 2022 ). Genetiierfstat  and adegierfstat ,55. Nei ierfstat .


However, patients that resumed belatacept/withdrew CNIs (n = 10) had a trend towards a better eGFR comparing with the others (n = 15): 54 ± 20 mL.1.73 m−2 vs. eGFR 43 ± 16 mL.1.73 m−2, respectively (p = 0.15). The only factor associated with belatacept resumption was when the withdrawal took place during the COVID-19 outbreak. Transcriptome analysis of PBMCs, did not support rebound in alloimmune response. Conclusions: These findings underpin the use of belatacept as part of a time-limited therapy, in selected kidney transplant recipients, possibly as an approach to allow efficient vaccination against SARS-CoV-2.Introduction: In kidney transplant recipients, belatacept is usually pursued indefinitely after it has been started. In the setting of the belatacept shortage and after having evaluated the benefit–risk ratio, we established a strategy consisting of time-limited belatacept therapy/transient calcineurin inhibitor withdrawal, whose results are analyzed in that study. Methods: We considered all the kidney transplant recipients that had been switched from conventional immunosuppressive therapy to belatacept and then for whom belatacept has been withdrawn intentionally. Furthermore, in the first 8 patients, we assessed changes in peripheral blood mononuclear cells (PBMC) transcriptome using RNAseq before and 3 months after belatacept withdrawal. Results: Over the study period, 28 out of 94 patients had belatacept intentionally withdrawn including 25 (89%) switched to low-dose CNI. One rejection due to poor compliance occurred. The eGFR after 12 months remained stable from 48 ± 19 mL.1.73 m During the last decades, improvements in long-term graft survival have been slight . From stWe carried out a monocentric study on all consecutive adult patients who underwent kidney transplantation between 1 January 2010, and 31 December 2019, at the Nantes University Hospital. Firstly, from all patients that have been switched to belatacept, we considered those for whom belatacept has been stopped during follow-up. Meaning that those deceased, returned in dialysis, with missing data and who remained on belatacept at the last follow-up we excluded. Secondly, we differentiated those for whom the decision to stop belatacept had been planned with the clear intention of time-limited belatacept therapy (see below population section of the results) and those for which the discontinuation was driven by other causes and excluded the latter ones.www.divat.fr, CNIL final agreement, decision DR-2025-087 [No914184] 15 February 2015).All data were extracted from the French, multicentric, observational and prospective DIVAT cohort of transplanted patients  and 250 mg bolus of methylprednisolone. The standard post-transplant immunosuppression includes calcineurin inhibitor (CNI), namely tacrolimus (trough level between 6 and 10 ng/dL) or cyclosporine  and mycophenolate mofetil  or mycophenolic acid .High-immunological risk patients (definite by panel-reactive antibody (PRA) > 75%) received induction immunosuppression with rabbit antithymocyte globulin  6 mg/kg and a 250 mg bolus of methylprednisolone followed by triple immunosuppression including CNI, MMF or MPA, and prednisone.Our standard protocol planned steroids withdrawal between 1 and 3 months, but some patients remained on triple therapy  or dual therapy with CNI and steroids in case of MMF/MPA withdrawal due to poor clinical tolerance and/or infections.After 2016, low-immunological risk patients received a low dose of rATG (3 mg/kg) as induction therapy instead of basiliximab.Conversion from conventional therapy to belatacept in some patients  was decided on an individual level at the discretion of clinicians, mainly in patients with poor renal function and suspected to have adverse events related to tacrolimus or cyclosporine exposure. Belatacept administration schedule was 5 mg/kg, repeated at 2 and 4 weeks, then every 4 weeks. In most cases, tacrolimus was tapered after belatacept introduction with the posology halved after 2 weeks followed by a complete withdrawal after one month.At belatacept discontinuation, other immunosuppressive drugs such as tacrolimus or sirolimus were resumed on the day of the last belatacept injection.Recipient characteristics collected were gender, age, number of previous transplants, initial renal disease, and renal replacement therapy, history of hypertension or diabetes, presence of anti- Human Leukocyte Antigen (HLA) antibody, and DSA before transplantation.Donor features were age, donor type . Baseline transplantation parameters included cold ischemia time, number of HLA A-B-DR incompatibilities, induction therapy, initial maintenance treatment, use of steroids and delayed graft function. Estimated Glomerular filtration rate (eGFR) was estimated using the Modification of Diet in Renal Disease (MDRD) Study equation . During Data collection stopped upon last known visit, return to dialysis, or death.t-test was used to compare quantitative variables. After having checked for normality assumption, one-way repeated measures ANOVA was used to analyze eGFR kinetic, pairwise paired t-tests were used for comparison between two time-point. Analysis was conducted with RStudio version 1.4.1106 .The estimated Glomerular Filtration Rate (eGFR) values were calculated using the Modification of Diet in Renal Disease (MDRD) equation and reported as mean (SD). Qualitative variables were analyzed using Chi-square or Fisher’s exact test. Student’s For the 8 first patients for whom we had planned belatacept discontinuation in the context of the belatacept shortage (see below ®). Total RNA was extracted from all samples with the TRIzol® isolation protocol followed by QIAGEN RNeasy Micro clean-upprocedure. RNA samples with a > 7 RIN score were used. For 3′ DGE profiling, RNA-sequencing protocol was performed according to our implementation of Soumillon et al. protocol [n = 26). The mRNA poly(A) tails were tagged with universal adapters, well-specific barcodes, and unique molecular identifiers during template-switching reverse transcription. Barcoded cDNAs were then pooled. 200 ng of cDNAs were amplified and fragmented using a transposon-fragmentation approach which enriches for 3′ ends of cDNAs . A library of 350–800 bp was run on a 100 cycles SP run on Novaseq6000 at GenoA IRS-un platform facility (Nantes). Samples were demultiplexed and aligned on the hg19 genome using the 3′ SRP pipeline.PBMC were isolated by gradient protocol . Conditions for the comparison corresponded to the D0 and M3. Gene ontology enrichment analyses were performed using Enrich [https://www.ebi.ac.uk/ena (accessed on 1 June 2021)), understudy accession PRJNA492956.The primary analysis of DGEseq data including, quality controls of reads, demultiplexing, read mapping, and quantification of gene expression, was carried out as described in Charpentier et al.  Normalizg Enrich . The samg Enrich . Raw RNA2, (ii) low immunological risk according to the judgment of their referring practitioner. In March 2020, the COVID-19 pandemic threatened to overwhelm the hospital’s capacities, and attendance at day-hospital  was considered unsafe, the implementation of the process was sped up.In the context of the belatacept shortage, a strategy of time-limited belatacept therapy was set out, that consisted of reconsidering its prescription in all patients that had been treated with belatacept for at least one year. Discontinuation and concomitant resumption of CNI or mTOR inhibitor at low dose was offered to patients that fulfilled the following criteria: (i) steady eGFR > 30 mL/min/1.73 mDuring the study period, 94 patients were switched to belatacept, then it was discontinued in 36 patients, including 28 for whom the decision to stop belatacept has been planned intentionally as outlined above . In 10 on = 25, 89%) were transplanted for the first time, mostly from a brain-dead donor  including 13 (62%) extended-criteria donor. Whereas induction therapy was predominantly antithymocyte globulin , initial maintenance therapy was almost always an association of tacrolimus , mycophenolate derivatives , and prednisone . Only 1 patient endured an acute rejection before belatacept introduction (active ABMR). Incidence of delayed graft function was 36% (n= 10).In total, 12 out of 28 (42%) were male, their mean age was 51 years, and most of them  or suspected CNI-related adverse events . In one case, the reason for the switch was an active ABMR. The switch timing was equivalently distributed between early , late  and intermediate  posttransplant. Except one, all received other immunosuppressive drugs: mycophenolate derivative , steroids  or both , only two patients continued a low dose CNI therapy either with tacrolimus or cyclosporin, for focal and segmental glomerulosclerosis relapse and ABMR before conversion, respectively  then steadily to reach 46 ± 12 mL.1.73 m−2 at 1-year post-conversion (p = 0.003).As illustrated in n = 17, with azathioprine n = 2, with mTOR inhibitor n = 2, alone n = 2).Belatacept discontinuation occurred at a mean time post-transplant of 41.2 (12.9–188.4) months when patients had been given belatacept for 21.6 (4.3–50.8) months. Except for three cases (two monotherapies with mycophenolate derivative and one with mTOR inhibitor), the new immunosuppressive regimen was based on tacrolimus . One patient (83 years old) died of an unknown cause at his residence, and one was lost to follow-up because of relocation, both shortly after belatacept discontinuation.n = 2), digestive disorders (n = 2), diabetes (n = 1)), patient request (n = 3), and finally decline of the renal function (n = 2). Belatacept was resumed in 10 out of the remaining 25 patients after a mean time of 5.1 (1–15.3) months A. Mentiop < 0.01); among them were all patients who had belatacept reintroduced at their request. No other parameter was associated with belatacept resumption including the eGFR at the time of the belatacept withdrawal or the type of immunosuppressive drugs that have been resumed.Remarkably, 8 out of the 10 patients that resumed belatacept had been discontinued at the time of the COVID-19 pandemic (−2 to 45 ± 15 mL.1.73 m−2 (p = 0.12). Afterward, the mean eGFR remained stable: 46 ± 17 mL.1.73 m−2 at 1-year post-discontinuation (p = 0.20) , in line with the reversible acute hemodynamic effect of the CNIs on the glomerular tuft. Three months after belatacept discontinuation and simultaneous resumption of other immunosuppressive drugs, a slight drop in eGFR was observed, from 48 ± 19 mL.1.73 m = 0.20) B. When w = 0.20) C. One-yeCNI-free immunosuppressive regimen using belatacept not only has been demonstrated to improve renal function but also to avoid CNI-related adverse events and decrease the development of DSA. As described in As already mentioned, after belatacept discontinuation, one patient experienced a severe rejection due to poor adherence. Except for this patient none developed either rejection or DSA.In the first eights patients for whom belatacept was withdrawn on purpose, who all resumed with CNIs, we assessed by RNAseq changes in peripheral blood mononuclear cells (PBMC) transcriptome before (D0) and after that event (1 month (M1), 2 months (M2), and 3 months(M3)). As shown in Our study reports for the first time the implementation of a time-limited belatacept therapy or in other words a transient CNI withdrawal mostly in patients with poor renal function in the early period post-transplantation. After the conversion from belatacept to another immunosuppressive regimen containing low dose CNI in most cases, we observed a slight drop in eGFR without subsequent change at 1-year post-conversion and did not notice either acute rejection/sensitization or infectious/metabolic adverse event. Remarkably, in patients in whom belatacept was eventually resumed/CNI withdrawn, the eGFR rapidly improved, arguing for a hemodynamic and reversible effect of CNI on the glomerular tuft. Finally, in a group of patients, changes in RNA-seq transcriptome profiles of PBMCs did not suggest a rebound of the alloimmune response after belatacept withdrawal.Nowadays, belatacept is increasingly used as part of a conversion strategy with the purpose to withdraw CNI in the increasing patient population with poor renal function a few months after transplantation. Studies reporting the outcomes of this strategy have constantly found an improvement of renal function, certainly caused by the relaxation of the glomerular tuft vasoconstriction in kidneys previously injured by the transplantation ,10,11,122 and the authors concluded that belatacept should not be stopped. However, in many cases, belatacept withdrawal occurred in the setting of acute complications, sometimes serious, making the interpretation of the results difficult. When the authors focused on the 13 patients for whom belatacept had been withdrawn apart from any complication, no significant change was observed in eGFR. Consistent with ours, this finding suggests that the conversion from belatacept to tacrolimus might be safe when accomplished on purpose, in patients with a suitable kidney function and with careful monitoring of the tacrolimus trough levels that should be maintained low around 6 ng/mL, enough when the immunological risk is low [Only one study has reported a multicentric series of 44 patients converted from belatacept to another immunosuppressive drugs . Resultsk is low .Beyond a drop in GFR, the other theoretical risk of changing immunosuppressive drugs is to favor acute rejection/allo-sensitization. We did not observe such an event except in one case, related to non-compliance. This case highlights that when belatacept is introduced to ensure efficient immunosuppression, as it has been proposed especially in young patients , it shoun = 2) and diabetes triggered by CNI (n = 1), and, importantly, all returned after the CNI withdrawal. In the others, causes were adverse events supposed to be CNI-related (n = 4) and at the patient’s request (n = 3).It is worth noting that 10 out of 25 patients resumed belatacept, which might be interpreted as a failure of our strategy. However, only 3 patients had it reintroduced for objective reasons, such as kidney function degradation (Only the timing of the belatacept discontinuation, namely when it had been implemented at the time of the COVID-19 outbreak, was significantly associated with belatacept resumption. We may assume that in this very specific setting, patients felt compelled to stop belatacept, explaining the high rate of resumption afterward. That highlights the importance of therapeutic patient education to ensure adherence to treatment change .Our study has some obvious limitations, mainly the low number of patients and the limited follow-up after the conversion from belatacept to other immunosuppressant drugs. Consequently, we cannot ensure that long-term kidney function could not have been impacted by CNI exposure. However, chronic CNI nephrotoxicity is increasingly questioned. In this regard, our finding that the expression of HCK, a gene found to be a key driver on kidney fibrosis , was sigOur findings raise the question of the potential benefit of using belatacept in a time-limited way, apart from the avoidance of iv injection and a potential medico-economic advantage. Except for the well-known risk of EBV-induced lymphoproliferative disease in seronegative recipients, and based on the data from BENEFIT ,6 along In all, our findings underpinned the concept of a time-limited belatacept therapy in a selected group of kidney transplant recipients. Further evaluation including well-conducted prospective studies with a long-term follow-up investigating the benefit–risk ratio of that strategy are needed.


The study of brain functional connectivity alterations in children with Attention-Deficit/Hyperactivity Disorder (ADHD) has been the subject of considerable investigation, but the biological mechanisms underlying these changes remain poorly understood. Here, we aim to investigate the brain alterations in patients with ADHD and Typical Development (TD) children and accurately classify ADHD children from TD controls using the graph-theoretical measures obtained from resting-state fMRI (rs-fMRI).We investigated the performances of rs-fMRI data for classifying drug-naive children with ADHD from TD controls. Fifty six drug-naive ADHD children  and 56 age matched TD controls  were included in this study. The graph measures extracted from rs-fMRI functional connectivity were used as features. Extracted network-based features were fed to the RFE feature selection algorithm to select the most discriminating subset of features. We trained and tested Support Vector Machine (SVM), Random Forest (RF), and Gradient Boosting (GB) using Peking center data from ADHD-200 database to classify ADHD and TD children using discriminative features. In addition to the machine learning approach, the statistical analysis was conducted on graph measures to discover the differences in the brain network of patients with ADHD.An accuracy of 78.2% was achieved for classifying drug-naive children with ADHD from TD controls employing the optimal features and the GB classifier. We also performed a hub node analysis and found that the number of hubs in TD controls and ADHD children were 8 and 5, respectively, indicating that children with ADHD have disturbance of critical communication regions in their brain network. The findings of this study provide insight into the neurophysiological mechanisms underlying ADHD.Pattern recognition and graph measures of the brain networks, based on the rs-fMRI data, can efficiently assist in the classification of ADHD children from TD controls. Attention-Deficit / Hyperactivity Disorder (ADHD) is one of the most common childhood psychiatric disorders. According to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) diagnostic criteria, ADHD is defined as inattention and/or hyperactivity/impulsivity. Children with ADHD may have problems concentrating on a specific task or sitting quietly without fidgeting for long periods. The most prominent ADHD phenotypes are ones with predominantly inattentive presentation (ADHD-I) and those that combine inattention and hyperactivity/impulsivity symptoms (ADHD-C) . ADHD tyToday, the combination of modern network science and neuroimaging techniques has made it possible to study the functional network of the human brain . The resIn recent years, graph approaches have also gained much popularity in studying the structural and functional connectivity of the brain at various scales for mapping human brain networks. Graph theoretical approaches provide a mathematical framework in which graphs comprise a set of nodes and edges. In the brain, nodes may correspond to predefined brain regions, whereas edges represent functional pairwise correlations among those nodes. While previous studies demonstrated the ability of rs-fMRI in identifying patients with ADHD from healthy controls, the utility of graph measures obtained from rs-fMRI data in the classification of ADHD from TD controls, has not been completely explored .Despite the remarkable success of machine learning approaches in rs-fMRI for disease diagnosis, automatic classification of patients with ADHD from typical development subjects using graph measures obtained from rs-fMRI data as interpretable biomarkers has received little attention in ADHD neuroimaging studies . FurtherSince neuroimaging evidence suggests that the characteristics of the small-world network are affected by neurodevelopment disorders, the optimal balance between global integration and local segregation may be disturbed during ADHD . Thus, w1 The ADHD database consists of eight different centers, making the data diverse and complex. Peking University was selected for this study because of the large sample size. It includes 143 TD subjects and 102 children with ADHD. A total of 112 right-handed subjects between the ages of 8 to 17, including 56 patients with ADHD, were selected from the Peking center according to the DSMIV-TR criteria and 56 typical development (TD) controls after applying the inclusion criteria.The present study used resting-state fMRI data obtained from the publicly available ADHD-200 Consortium.via the International Neuroimaging Data-sharing Initiative (INDI) are fully anonymized by HIPAA Privacy Rules.Psychiatric diagnoses were verified at Peking University through psychiatric interviews with experienced child psychiatrists using the Schedule of Affective Disorders and Schizophrenia for Children–Present and Lifetime Version (K-SADS-PL) administered to parents and children, and the ADHD Rating Scale-IV. Intelligence was evaluated with the Wechsler Intelligence Scale for Chinese Children-Revised (WISCC-R). The following exclusion criteria were applied to all participants: (1) a history of conduct disorder, oppositional defiant disorder, Tourette’s disorder, and any neurologic disorder that could impact the functional connectivity patterns of the brain; (2) a score of less than 80 for the full-scale WISCC-R; (3) a mean frame-wise displacement (FD) more than 0.3 mm; and (4) missing even one of the clinical phenotype listed in High-resolution whole-brain T1-weighted 3D MPRAGE data were acquired for each participant on a SIEMENS TRIO 3-Tesla MRI scanner with the following imaging parameters: repetition time (TR) = 1700 ms, echo time (TE) = 3.92 ms, field of view (FOV) = 256 mm × 256 mm, 176 slices, thickness/gap = 1.0, and voxel resolution = 1.0 mm × 1.0 mm × 1.0 mm; flip angle = 12°. The rs-fMR imaging was obtained axially using a blood oxygenation level-dependent contrast sensitive gradient echo-planar imaging , 220 mm × 220 mm (FOV), 64 × 64 (resolution), 30 slices, 4.5/0 mm thickness/gap. During the acquisition of functional data, participants were asked to relax with their eyes close and not to concentrate on anything in particular.Resting-state fMRI data preprocessing was carried out using the GRETNA toolbox and SPM12 . The firThe overall procedure of the current study can be seen in To create a 116 × 116 symmetric correlation matrix for each subject, Pearson correlation was calculated between each pair of regional time series. Finally, to explore the properties of brain functional connectivity , all the correlation matrices were thresholded into a binary graph with a fixed density level for further graph theory-based analyses, where nodes stand for brain regions and edges represent undirected connections between nodes. Fixed network density is the ratio of the actual number of edges divided by the maximum feasible number of network edges. Thus, this thresholding approach makes it possible to investigate the relative network organization by imposing on each network the same number of edges or wiring costs for compensatory compatibility. Since there is no specific method for selecting a unique threshold, we utilized a wide range of cost values  in steps of 1%.The whole-brain functional network  analysis was conducted using the GRETNA toolbox . Graph tTo further explain the characteristics of the small-world network, some parameters such as gamma and sigma were defined. Networks with small-world properties have a relatively high gamma, which is defined as follows:Also, these networks have almost the same Lp compared to random networks. Therefore, the sigma in the networks with small-world properties becomes more than one.Graph theoretical approaches can provide valuable insights into network efficiency in terms of how information is transferred across a network. Local efficiency indicates the information flow transmission in a network at the local level. It provides insight into the segregated ability of the brain network . ConsequUsing a feature selection algorithm is the necessary part of a machine learning technique, which promotes data interpretation, and subsequently improves the performance of a classification system. Recursive Feature Elimination (RFE) is a robust algorithm for feature selection presenting a precise way to determine the meaningful features before using them as input for a machine learning algorithm. RFE uses all features to construct a classification model. Then, it ranks the cooperation features in the classification model into a ranked feature list. Finally, RFE eliminates the irrelevant features that have a senseless collaboration with the classification model .The classification of ADHD children from TD controls was conducted using three different classifiers, including support vector machine (SVM), gradient boosting (GB), and random forest (RF). Then, these supervised machine learning algorithms were trained using a set of input data to yield the desired output. Since the obtained classification accuracy by SVM with linear kernels was similar to that of non-linear kernels and considering that solving the optimization problem for SVM with the linear kernel is much faster than the non-linear kernel, in this study SVM with a linear kernel was used. The number of available data in imaging research is usually low due to the high cost of data acquisition. Therefore, different cross-validation methods have been proposed to overcome the loss of generalization resulting from the small training and testing sample size. Since age, sex, race, handedness, and imaging parameters can be potential confounding variables in examining subjects’ brain network topology, we selected training and testing data only from the Peking center to not only classify drug-naïve ADHD children from TD controls but also more accurately investigate the functional brain changes in ADHD children.The k-fold cross-validation strategy presents a trade-off between bias and error variance. Therefore, selecting optimal values for “k” ensures that the selected method provides a suitable estimate of the model performance. If the value of k is small, cross-validation can lead to a lot of bias for error. The k-fold cross-validation with a high value of k can lead to a lot of error variance and is computationally intensive. In machine learning scenarios, the value of k is usually used between 5 and 10. In the present study, the best accuracy was achieved using k-fold cross-validation of 9. The procedure is iterated until all subjects are considered once as the test sample. Eventually, the results of all repetitions are averaged to provide the ultimate classification accuracy .Using the data of one center as training and test data may reduce the generalizability of a classification model and cause inflation of accuracy in the test data due to an error of including all data when selecting features for the classification model. For the classifier to be insured free of circular analysis, we used an iterative cross-validation procedure where the feature selection (RFE) step was performed using the GF model only on the training set of every iteration.t-tests. Group differences in small-world properties were assessed applying ANCOVA, with age, sex, FD, and IQs as covariates. Throughout, multiple comparisons were controlled using the false discovery rate (p < 0.05).Statistical analyses were performed in SPSS 16.0. For each continuous variable, normal distribution was assessed by the Shapiro-Wilk test. Statistical group differences between the ADHD children and typical development children in age, sex, and all type of IQs were determined using T = 0.920, p > 0.05), a lower mean IQ , more males  and more head-motion . There were no significant differences in age and FD between the two groups, as detailed in As mentioned above, –3, FDR corrected)  and ], and thrrected) . HoweverDifferent classifiers, such as RF, SVM, and GB have been used in many machine learning based neuroimaging studies. We investigated the performance of these classifiers for discrimination of ADHD children from TD controls. The best performance was achieved using the gradient boosting classifier. The performance of the classifiers was compared by employing a nine-fold cross-validation strategy in p < 0.032). The global efficiency  of the ADHD group was significantly lower, whereas local efficiency was significantly higher than that of TD controls (p < 5.7 × 10–5). Locations of the AAL brain regions that show highest discrimination ability corresponding to the optimal subset of graph measures are shown in The top twelve features corresponding to the best classifier (GB classifier), are listed in The hub nodes were determined in two groups of children, i.e., ADHD, and TD. Nodes with a betweenness centrality of two standard deviations higher than the average of the betweenness centrality of all nodes were identified as hub regions. As listed in The small-world characteristics of functional networks of the human brain have been confirmed by different imaging modalities such as EEG, MEG, and fMRI . The curWe found that the characteristic path length in ADHD group was longer than in the TD group , which iAlso, we used resting-state fMRI and combined the graph-theoretical approach with the machine learning methods to classify drug-naive ADHD children from typical development (TD) controls. We used three different classifiers  with a small number of graph measures selected by a recursive feature elimination algorithm and achieved high accuracy in classifying ADHD children from TD controls. Our results revealed that the gradient boosting classifier outperformed the two other classifiers in terms of accuracy, sensitivity, and positive predictive value . The higThe GB is an ensemble of the decision tree model. First, a simple model is created on the data then the model error is evaluated. If the error was not reduced enough, another model is added to the old model to decrease the error. This operation continues until the desired error is obtained. Therefore, the gradient boosting algorithm can learn complex patterns . In RF cThe top 12 graph features with the most discrimination capability in Studies on various brain disorders have revealed that brain lesions are mostly located in the hub areas, hence investigating the hub areas may be a suitable approach for understanding brain disorders .As listed in Despite all the strengths of this work, there are several limitations to this study should be further addressed. First, we used the structural atlas AAL 116, which is common in functional studies. Since there is no one-to-one correspondence in the definition of functional and structural brain areas, using a functional atlas such as Power 264 atlas can be a more suitable option. Second, we used the binary graph for brain network construction. However, the use of the weighted graph and its unique features may be effective in distinguishing between ADHD and TD controls, as well as providing information on the physiological mechanism of ADHD. Third, we only used the rs-fMRI data in our study. Using multi-modal imaging  and non-imaging  data may provide additional information that can better characterize the discrepancies between ADHD children and TD controls. Fourth, since graph theory measures hold potential of clinical significance, they deserve further study in neuroimaging research in the areas of diagnosis, prognosis, and psychopharmacology and therapeutics of ADHD.We combined the binary graph features obtained from rs-fMRI data with machine learning methods to classify drug-naïve ADHD children from the typical development (TD) controls. Our results showed that in ADHD children, regions located in the frontal lobe  undergo changes more than other brain regions. We determined optimal graph features with high discrimination ability using a recursive feature elimination algorithm to provide insights into ADHD pathophysiology. The classifiers were trained and cross-validated, then their performances were evaluated. We found that the best classifier for classifying ADHD children from TD controls obtained an accuracy of 78.2%. We also performed a hub node analysis and found that the number of hubs in TD controls and ADHD children were 8 and 5, respectively, indicating that children with ADHD have the disturbance of critical communication regions in their brain network. These graph features facilitated machine learning algorithms to accurately classify ADHD from TD controls, indicating that graph features should be given more attention as potential biomarkers in neuroscience studies. This study brings an original methodological approach to advance the search for reliable and accurate biomarkers for the diagnosis of ADHD. However, the findings are still far from providing clinical advantages, and there is still uncertainty about the reliability of potential neuroimaging biomarkers for ADHD diagnosis. Therefore, further methodological, neuroscience and translational clinical neuroscience research is needed.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.This study involving human participants were reviewed and approved by the Research Ethics Committee of School of Medicine Mashhad University of Medical Sciences. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.MR and PH conceived the original idea. MR and SN carried out the experiment. HZ and HH supervised the project. SN wrote the manuscript with support from MR. All authors contributed to the article and approved the submitted version.


Direct and indirect smoking exposure is highly related to mental health in children. This study aimed to identify the association between exposure to smoking or secondhand smoke (SHS) and attention deficit hyperactivity disorder (ADHD).We used data from the Korean National Health and Nutrition Examination Survey (KNHANES) from 2007 to 2019, including variables such as diagnosed ADHD, smoking status, SHS, and urine cotinine levels among children and adolescents. We estimated the risk of ADHD according to smoking or SHS exposure in various exposure groups using adjusted logistic or linear regression models.Among 16434 participants, 133 children were diagnosed with ADHD (0.8%). Of these, 58 (43.6%) were aged <12 years and 75 (56.3%) were aged ≥12 years. Smoking was significantly associated with ADHD . SHS exposure and ADHD were attenuated after adjustment  in the logistic regression model. Smoking history was statistically associated with a younger age of ADHD diagnosis in the linear regression model.Smoking and the amount of smoking among children and adolescents was associated with ADHD. The Diagnostic and Statistical Manual of Mental Disorders (DSM) criteria are used to diagnose ADHD based on the symptoms and rating performance for diagnosis acquired by the teacher and parents or through a self-questionnaire. The overall prevalence of ADHD is 3–8% and is dependent on different regions: 6.2% in North America, 4.7% in Europe, 3.7% in Asia, and 2.3% in the Middle East2. In Korea, the prevalence of ADHD was reported variously by data or the type of study4. A current report indicated the rate of diagnosed ADHD treated with medications was 0.8% in South Korea5. Additionally, the prevalence of each type of ADHD differed6. Therefore, aberrant prevalence rates indicate various factors that influence ADHD.Attention deficit hyperactivity disorder (ADHD) is a neurobehavioral disorder characterized by hyperactivity, impulsivity, and inattention in children and adolescents7. Among the various factors, smoking was highly associated with the health burden of ADHD in children and adolescents9. The longitudinal effect of tobacco smoking may affect frontal cortical thickness10. Structural MRI studies of ADHD have revealed volume reduction of cortical thickness abnormalities in the frontal and parieto-temporal regions11. An animal model randomly exposed to tobacco smoke had neuronal cell damage and a decreased brain surface12. However, the question of whether smoking influences ADHD remains.Most studies analyzing the risk factors for ADHD assessed genetic inheritance and biological or environmental factors14. The United Kingdom prospective cohort study revealed the association between maternal smoking during pregnancy and child ADHD15, and the Danish National Birth Cohort indicated that prenatal nicotine exposure might have a causal role in ADHD16. A Finnish nationwide report revealed a dose-response relationship between nicotine exposure during pregnancy and offspring ADHD17. However, the Norwegian mother and child cohort study suggested that prenatal maternal smoking is not firmly related to offspring ADHD compared to paternal and grandmother smoking18.Prenatal smoking increases the risk of ADHD19. The National Survey on Children’s Health in the US found that secondhand smoke exposure is associated with childhood neurobehavioral disorders, including ADHD20. The Korean version of the study of ADHD rating scales reported that SHS in children is associated with a decline in neurocognitive performance21. Nevertheless, the association between SHS exposure and ADHD remains unclear22. Thus, the causality of these inverse associations needs, is clarified.Exposure to environmental tobacco smoke (SHS) is associated with a health burden on children’s organ systemsUsing data from the Korean National Health and Nutrition Examination Survey (KNHANES) from 2007 to 2019, we investigated the risk of related factors of ADHD among Korean children and adolescents, focusing on smoking status and SHS exposure.23. As data collection was performed by highly skilled surveyors and controlled for quality, the data were considered highly accurate and reliable23. All KNHANES participants provided written consent to participate in the survey and consent to the use of their personal data. The KNHANES data are openly available in a public repository (https://knhanes.kdca.go.kr/knhanes/eng/index.do).The Korea National Health and Nutrition Examination Survey (KNHANES) is a series of nationally representative, cross-sectional, population-based surveys of Koreans’ health and nutritional status conducted by the Korea Centers for Disease Control and Prevention. This annual survey uses a stratified and multistage probability sampling design to select household units, including non-institutionalized Korean civilians. Approximately 200 geographical sampling units from 17 central provinces of the Republic of Korea were used in the KNHANES 2007–2019. In total, 4000 to 5000 households are selected for participation in the survey annually. All family members respond to the questionnaires individually. The response rate ranges from 78.4–81.9% each yearThe current study used KNHANES (2007–2019) survey data, which included information on SHS, urine cotinine sampling, smoking history, and ADHD-related history in children and adolescents. Of the 105732 participants initially enrolled, we excluded those aged ≥19 years and those with missing data or refusal to provide data on age, sex, household income, self-rated health status, and smoking experience; and finally, 16434 participants were included. We divided the dataset into three analysis groups based on the KNHANES data design: Group 1 had 3212 participants with information on SHS exposure; Group 2 had participants with urine samples for urine cotinine levels; and Group 3 included participants with information on smoking history.ADHD was confirmed in children and adolescents with physician-diagnosed ADHD who responded ‘yes’ to the healthcare interview question: ‘Have you ever been diagnosed with ADHD by a doctor?’. Children and adolescents with physician-diagnosed ADHD were also asked about their age at diagnosis using the following question: ‘If yes, how old were you at the time of the diagnosis?’.The questionnaire contained questions on smoking experiences during their lifetime: ‘Have you ever smoked a cigarette?’ was used to define the smoking experience. The exposure to SHS at home during the day was defined with a ‘yes’ response to the following question: ‘Is there any person who smokes routinely inside the house?’. Participants were also asked about the number of smoking days and number of smoked cigarettes per day during the last month with the following questions: ‘In the past month, how many days did you smoke at least one cigarette?’ and ‘On average, how many cigarettes per day did you smoke in the past month?’. Smoking history was calculated using pack-days from the responses to the questions above.24. The German External Quality Assessment Scheme (G-EQUAS) was used to measure urine cotinine levels as part of external quality assurance and control.Urine samples were collected from the selected participants by a trained pathologist using a urine specimen cup. The participants were requested to submit early morning samples whenever possible. All samples were stored in an icebox, maintained at 4–7°C, and transported to the authorized central laboratory. For the cotinine analysis, 175 μL diphenylamine, the internal standard, was added to the urine samples, then 1 mL of each urine sample was hydrolyzed with 50 μL of 0.1M sodium hydroxide and extracted with 500 μL of chloroform. After centrifugal separation , the residue was dried using sodium sulfite. Urinary cotinine (ng/mL) was analyzed by gas chromatography–mass spectrometry using a PerkinElmer Clarus 600T instrument Baseline characteristics, such as age, sex, and household income, were used in the current study. Household income was assessed in quartiles. Children and adolescents were asked to self-rate their health which was considered a confounding variable.Statistical analyses were conducted using the SAS statistical software . ADHD status was calculated using the chi-squared test based on the baseline characteristics and smoking exposure status. A logistic regression analysis investigated the association between direct and indirect smoking exposure and ADHD. Direct smoking exposure was estimated based on smoking experience, and indirect smoking exposure was defined as SHS exposure. Crude and adjusted logistic regression models were used in this study. The logistic regression model was adjusted for age, sex, household income, and self-rated health status. The adjusting factors were selected in a stepwise backward manner. Linear regression models were developed to demonstrate the dose-response relationship between direct/indirect exposure to smoking and age at ADHD diagnosis. Direct smoking exposure was estimated by smoking history calculated by pack-days in the last month, while indirect smoking exposure was defined as the urine cotinine level, a continuous variable. Adjusted linear regression models were developed after adjusting for age, sex, household income, and self-rated health status; a two-tailed p<0.05 was considered statistically significant for all statistical calculations.We retrieved data from 105732 participants of the 2007–2016 KNHANES and excluded 89298 participants because they were aged ≥19 years and had missing or refused to provide data. Therefore, 16434 participants aged <19 years were included in this analysis and divided into three groups. Analysis Group 1 included 3212 participants with data on SHS, analysis Group 2 had 5499 participants with data on urine cotinine levels, and analysis Group 3 included 521 participants with a smoking history within the last month .Of the total participants, 133 were diagnosed with ADHD (0.8%), 58 (43.6%) were aged <12 years, and 75 (56.3%) were aged ≥12 years. A greater proportion of the children with ADHD were males (M:F=99:34), and the second quartile of household income was more prominently distributed in the non-ADHD and ADHD groups. In all, 1081 participants reported a smoking history, and 16 had ADHD. In the analysis Group 1, 1672 participants answered that they were exposed to SHS, including 14 children with ADHD. In analysis Group 2, the group with ADHD  had higher urine cotinine levels than the group without ADHD . In analysis Group 3, of the 521 participants with information on smoking history, the amount of smoking was higher in the ADHD group (17.33 pack-days) than in the non-ADHD group (8.18 packdays) .The results of the logistic regression model showed that smoking experience was significantly associated with ADHD . However, the association between SHS exposure and ADHD was attenuated after the adjustment  .Linear regression models based on the age at diagnosis of ADHD and smoking history or urine cotinine level, smoking history, and urine cotinine level were statistically correlated with the age at ADHD diagnosis. The higher the number of cigarettes smoked, the younger the age at ADHD diagnosis  .The current study investigated the association between ADHD, smoking experience, and SHS exposure among Korean children and adolescents and found that smoking exposure was closely associated with ADHD. The age at ADHD diagnosis was significantly younger among adolescents with a higher smoking experience than among others. SHS exposure and urinary cotinine levels were related to ADHD; however, these were attenuated after adjustment.26. The progression of ADHD was quickly aggravated in young patients with ADHD with smoking experience, and cravings were much more severe while quitting smoking28. A 16-year longitudinal study reported that the group with ADHD had higher rates of daily smoking and more severe withdrawal symptoms than the non-ADHD children and adolescent groups27.Clinically, smoking is closely related to ADHD in young people who had ADHD. Adolescents who tended to smoke more had increased symptoms of ADHD, such as inattention or hyperactivity29. Animal models in which the dopamine transporter gene was knocked down or knocked out exhibited an ADHDlike phenotype, which increases activity levels that normalize to wild-type levels with stimulant administration22. Smoking also increases dopamine production, associated with dopamine release, through accelerated atrioventricular conduction30. The nicotine-dependent human brain is also involved in neuronal processing-mediated working memory31. Smoking, which shares a similar mechanism with ADHD, results in adverse outcomes on human traits of executive function.Several mechanisms are responsible for how smoking results in ADHD. The suggested theory of ADHD pathophysiology is associated with dopamine dysfunction, which leads to disrupted reinforcement resulting in executive dysfunction32. Zubair et al.33 reported that children exposed to SHS at home had significantly increased odds of having more than two neurobehavioral disorders, including ADHD, learning disabilities, and conduct disorders, compared to those not exposed to SHS20. A teacher-rated rating scale in Korea confirmed that SHS exposure in children is associated with neurocognitive dysfunction such as inattentiveness and hyperactive performance21. SHS exposure increases executive function problems in rodent models33. A large population-based study reported that paternal smoking during pregnancy increased the risk of ADHD34. Our data support the hypothesis that the association between SHS exposure and ADHD is statistically significant, with a need for follow-up studies.We also found that SHS exposure was associated with a higher risk of ADHD, even though the statistical significance was attenuated after adjustment. SHS exposure and infant and young children’s health are associated with increased respiratory tract infections, neurocognitive problems, and adolescent smoking35. Brain maturation is not completed before puberty, and human prefrontal cortex conducting executive function reaches stability at approximately 12 years of age36. Early exposure to nicotine interferes with cognitive development and leads to an executive deficit37. Young people with a previous ADHD history tend to start smoking early38. Moreover, ADHD is difficult to diagnose at an undisrupted daily life stage. Exposure to smoking in preschool children before ADHD diagnosis may increase the risk of ADHD expression compared with non-smokers39.Our study showed that the amount of smoking is related to the diagnosis of ADHD at an early age. Thus, age and nicotine exposure may influence executive brain function40. Young smokers may have a riskier decision than non-smokers because of immature inhibitory control41. Additionally, youths exposed to smoking from family members, siblings, and relatives may be at a greater risk of developing a nicotine habit than those without smoking exposure43. Voluntary smoke-free home policies can reduce the environmental exposure range in children by 20–50%44. Therefore, the smoking history of family members may influence potentially causing ADHD in children and adolescents, necessitating education and counselling on smoking cessation and abstinence in their families.Although the statistical significance was attenuated after adjustment, our results showed that urine cotinine levels were also associated with the risk of ADHD and the age at ADHD diagnosis. In an experimental mouse model, exposure to cigarette aerosols during the early life stage (detected through urine cotinine levels) altered frontal cortex developmentThis study has several strengths. To our knowledge, this is the first study showing the relationship between ADHD and smoking experience with detailed information on smoking, such as urine cotinine levels, period of smoking, and SHS exposure, using data from 2007 to 2019 of the KNHANES survey which was reliably designed and conducted as a governmentled nationwide survey with elaborately sampled participants.However, the study design may have limited the findings of this study. The KNHANES was a crosssectional study, and the interpretation of the causal effect of smoking on ADHD was limited. Nevertheless, we conducted several analyses using different definitions of tobacco exposure. We demonstrated an association between SHS exposure and ADHD or urine cotinine levels and age at ADHD diagnosis, even with an attenuated association after adjusting for a limited number of participants. The KNHANES collected data based on questionnaires, omitting confounders, such as genetic factors. In addition, the laboratory results showed a short exposure period because urine cotinine has a short half-life, and the smoking history only described events during the last month. Thus, further longitudinal studies with detailed information are needed to establish a relationship between ADHD and smoking.The results of this study demonstrated an association between smoking and ADHD. Smoking status and high and indirect exposure among children and adolescents are associated with ADHD. Additional studies on the prevention of smoking exposure in infants and young children are warranted.


Most studies on climate change’s impacts on agriculture focus on modeling techniques based on large-scale meteorological data, while few have investigated how farmer’s perception of climate change’s impacts can affect crop diversity and crop management practices, especially in industrialized contexts. To fill this gap, we conducted 24 semi-structured interviews in a study site located in the Catalan Pyrenees. Our results show for the first time in an industrialized context that farmers perceive multiple interrelated climate change impacts on local agroecosystems. For instance, snowfall and freeze events have decreased, which respondents associated with the increase of pests and diseases affecting both wild flora and cultivated plants. Similarly, changes in precipitation patterns lead to a perceived decrease in useful rain for agriculture. Farmers are also reporting changes in their management practices, such as increased irrigation or use of pesticides, which respond to these climatic factors but also to changes in the crops that are cultivated. Crop diversity is in decline in the area both at the species and landrace levels, especially in rainfed fields. This is mainly driven by socioeconomic factors such as agricultural abandonment or access to commercial seeds, although climate change factors such as increased pests or decreased rainfall can have an impact. Despite the crop diversity losses found, many landraces have been maintained, mainly due to their cultural value, and also new crop species have been introduced, which are now viable due to the increase in temperature. Although we focused on a specific case study, we found several trends that are also present in other contexts. Therefore, the results of this research are relevant at a global scale since they show that climate change is affecting mountain agroecosystems in industrialized contexts and may affect more drastically both agrobiodiversity and crop management practices in agroecosystems worldwide.The online version contains supplementary material available at 10.1007/s13593-022-00806-3. Crop diversity, or the variance in genetic and phenotypic characteristics of plants used in agriculture, is the result of millennia of favorable trait selection, aiming at producing larger, earlier ripening, culturally appropriate, or frost-resistant crops  in a study site in the Catalan Pyrenees. The site was selected because of its relative isolation  and the fact that farmer communities there have had long-term interactions with the environment. Our main objectives were 1) assess the perceived climate change impacts in the study area, (2) explore how farmers have changed their crop management practices to adapt to those impacts, (3) document changes in crop species and landraces, and (4) identify the climatic and socioeconomic factors driving those crop changes. To narrow down the scope of the study, we focused on the home garden agroecosystem when asking about changes in crop management and landraces . The subalpine zone is between 1600 and 2300 masl and is dominated by coniferous forests, mostly mountain pine and firs (Abies sp.). It is the most sensitive habitat to human action as it has been intensively exploited to supply coal and wood. The montane zone occupies the bottoms of the valleys, and it is composed of deciduous forests and riparian trees that follow the river’s courses such as oak, ash, birch, poplar, alder, or willow. Human presence has altered these ecosystems converting them into mowed meadows to feed livestock. However, the gradual abandonment of traditional livestock has recently triggered the expansion of forest cover  contributed to maintaining an agrarian economy based on self-sufficiency , participant observation , and a field diary to understand the context of the site, the perceived local indicators of climate change impacts (LICCI), the changes in crops, landraces, and management practices over time and the drivers and consequences of these changes.Interviewees were selected based on purposeful and snowball sampling, targeting elder farmers (60+ years old) with long-term experience and close relationships with (a) the local environment in general and/or (b) the home garden agroecosystem. Specifically, 22 home gardeners (17 women and 5 men) were interviewed (managing a home garden for at least 25 years), and two elders that, despite not having a home garden, had sufficient long-term knowledge about the local environment to be able to perceive climate change impacts and general changes in cropping systems. The sample’s gender balance is biased towards women because, in this area, women have historically been in charge of home gardens and seed management.Interviews (that lasted about one hour each) were digitally audio-recorded after obtaining agreement from participants . Interviews were conducted directly in the home gardens or at the interviewee’s houses but always following the LICCI project safety and security measures adapted to the COVID-19 pandemic. The interviews were performed following a script with different sets of questions, and they were conducted in Catalan . This script was based on the data collection protocols of the LICCI project and included questions such as “Have you perceived any change in the local environment ? And in the cropping systems in the area? If so, what is driving this change? And have you done anything to adapt to those changes?”.To analyze changes on a temporal scale, the interview questions included a relationship to the past, referring to when the interviewee was young and to specific historical events that were known . In cases where the interviewee was clear about the time of the observation, he or she was asked specifically about the year in which the change occurred. Moreover, a catalogue of landraces  and the total number of respondents (24).After data processing and analysis, we conducted five follow-up interviews with people from the same sample to clarify questions and resolve contradictory information.All of the interviewees indicated at least one perceived change in the climatic, physical, biological, or socioeconomic systems over time that were directly or indirectly impacting the local agroecosystems, primarily the home gardens , although respondents also reported changes in the physical, biological, and socioeconomic systems (see Table The most affected subsystem was the cryosphere (ice and snow), and the most mentioned change pointed to the decrease in the amount of snowfall (15 respondents). Changes in the frequency of freeze events and the duration and temporal distribution of snow cover were also reported. An elder home gardener from Lladrós town stated, “It used to snow much more than it does now. Grandparents celebrated the snow in November and December because it meant that in summer the water from the springs would come down. Now it snows less and there is less water in general”.More than half of the respondents mentioned changes related to rainfall. The most reported change in this subsystem was the fact that it used to rain more than now (12 respondents), both at the average general rainfall level and in a particular season (in spring and summer). Moreover, six respondents said that now the rain is more variable or harder to predict. Nine interviewees also mentioned that this has a negative consequence for the yield of crop fields and home gardens. An elder farmer from Lladorre stated, “It may fall the same amount of water as before, but nowadays there are strong storms, and the water is not as useful as in the past. Before, rainfalls were more regular and predictable; the rain was light so that the water could soak into the soil”. Reports of temperature changes (13 respondents) were also abundant and were mostly related to mean temperature increase (eight respondents) and an increase in temperature extremes (four respondents).Another much-mentioned observation was the change in the transition between seasons. Eight interviewees observed that in the past, seasons were more defined and transitioned more gradually than now.Regarding the terrestrial wild fauna subsystem, two respondents stated that now the mortality of insects has increased, and one interviewee mentioned that bees before were more abundant. Respondents associated this imbalance in insect populations  with changes in the terrestrial wild flora subsystem. Specifically, respondents stated that trees abundance and productivity have decreased, while mortality has increased. The decrease in the abundance of insects may be also due to an increase in the use of insecticides according to home gardeners.Despite this general trend of decrease in the abundance of insects, there is a perceived increase in pests affecting both wild trees and cultivated plants. Local perceptions associate this trend with climate change, as a home gardener from Cassibrós stated “Now there are more pests than before because in winter is not as cold and it snows less; therefore pests can survive easier”. Indeed, the most mentioned impact in the socioeconomic system, specifically to the agriculture subsystem, is the increase in crop diseases and pests, mentioned by 13 respondents. Home gardeners reported two major drivers of this increase. On the one hand, as snowfall and freeze events have decreased and temperatures have risen, pest populations have grown faster. Crop diseases follow a similar pattern particularly in the case of pathogens like fungi. Therefore, the spread of crop diseases and pests is linked indirectly with climate changes. On the other hand, interviewees also believe that this increase in pests and diseases is due to the decrease of beneficial insects for pest control such as ladybugs. For example, a respondent from Ribera de Cardós stated, “There used to be more ladybugs, bees, earthworms…These animals are good for home gardens since they prevent certain pests such as aphids”. Moreover, two respondents also mentioned that now there are many birds in the home gardens and fruit trees. The respondents’ explanation is based on the fact that there are no grain fields now where the birds could supply their feeding needs and therefore birds search now for additional food in home gardens. Finally, another perceived change in the agriculture subsystem includes changes in crop flowering time, which respondents relate to changes in precipitation and temperature patterns.There has been a notorious change in cropping systems and management practices in the valley. The landscape has changed drastically as a result of crop abandonment and depopulation as well as due to socioeconomic transformation. In the early 1960s, the change of self-subsistence economy to a market economy began resulting in a rural exodus from the valley and a consequently gradual increase of the forest mass.In parallel with this, respondents mentioned seven changes in management practices in home gardens directly or indirectly related to climate change , which in the past occupied large extensions of the valley, are no longer cultivated because nowadays, it is more profitable to buy the grain than to grow it. This is ultimately due to both the easier accessibility to the market and to the complicated orography, which makes the mechanization of agriculture in this valley difficult. Similarly, farmers used hemp for spinning fabrics and gave it to chickens and chicks. However, farmers no longer conduct these practices.Moreover, some crop species such as sunflower, thistle, white beet, and potato have been abandoned because their use was related to specific livestock that has been lost. For example, sunflowers were given as fodder to dairy cows, which was a traditional agrarian activity in this valley that was intensified from 1932 onwards, when the first dairy cooperative was created. Nowadays, milk production is not profitable in the valley because feed for cows, transportation, and labor costs have increased. Therefore, most cows are beef cattle today, thus, no protein-rich fodder crops are required since cows are left in the pasture lands. In line with this, thistle was used as rennet for cheese production, but now, it is no longer produced in the valley. In addition, white beets were used for pigs’ consumption, but pig farming has been totally abandoned.As explained so far, the abandonment of crop species is mostly due to socioeconomic changes. However, respondents also mentioned a potential climatic driver, the decreased rainfall, and the shift from rainfed cropping systems  to irrigated home gardens. As mentioned before, eight farmers stated that now it would be impossible to grow these crop species without irrigating them, and thus, decreased rainfall might be a potential underlying factor encouraging their abandonment.In contrast with this crop species loss, there are seven vegetables and fruits that have been introduced, specifically in the home gardens: eggplant, cucumber, melon, flat peach, red beet, watermelon, and pepper. These species have been introduced due to dietary changes and access to commercial seeds and seedlings. Apart from these socioeconomic drivers, three respondents stated that the reason for the introduction of these species is also climatic changes that allow farmers to plant some species that would not have grown in the past see Fig. . One of As per the landrace diversity, a list of 59 landraces cultivated by more than one generation has been obtained from the fieldwork  along with the changes associated with the use of these landraces, the drivers of those changes, and the influence of climate change impacts.Respondents mentioned eight landraces that are no longer grown and have therefore been lost. Moreover, many of the landraces still present in the home gardens are declining as the number of farmers who grow them is much lower than in the past. In fact, 25 landraces  have suffered a decrease in their use in home gardens of Vall de Cardós, leading to a loss of crop diversity. However, almost half of the landraces mentioned (45.9%) do not present any change or have increased their presence in home gardens.tomàquet del país (tomato landrace). Other reasons are problems of transportation (as in the case of préssec blanc peach tree landrace), or processing difficulties .The loss of landraces is due to several reasons. First, now people buy commercial seeds because it involves less labor work while in the past home gardeners sowed all the seeds themselves. For instance, an interviewee from Lladrós stated “Before, everything was seeded at home and now people buy seeds and plants because it is easier”. Therefore, one important driver of landraces’ changes is greater market accessibility. Moreover, some landraces have fallen into disuse because their seeds are not for sale, so people cannot access them unless they have someone that gives them seeds or they save their own seeds from one season to the next one. This is the case of . Therefore, one important driver of landraces’ changes is greater market accessibility. Moreover, some landraces have fallen into disuse because their seeds are not for sale, so people cannot access them unless they have someone that gives them seeds or they save their own seeds from one season to the next one. This is the case of tomàquet del país (tomato landrace). Other reasons are problems of transportation (as in the case of préssec blanc peach tree landrace), or processing difficulties .The loss of landraces is due to several reasons. First, now people buy commercial seeds because it involves less labor work while in the past home gardeners sowed all the seeds themselves. For instance, an interviewee from Lladrós stated “Before, everything was seeded at home and now people buy seeds and plants because it is easier”Second, and related to the first point, some landraces are very pest sensitive, and therefore, they are not effectively adapted to the new climatic context with pests increase. As an example, a woman from Lladorre said that “Here, there used to be a giant perera de Cardós (pear tree) that always produced a lot of pears, but now this type of pear tree is disappearing due to pests”. Since there are not as many inhabitants as in the past, certain crop species and trees are not cared for, and these landraces are gradually being lost. A home gardener from Ribera de Cardós stated that “Nowadays, as fewer people are living in the area, fava beans are sowed less and are being lost”.bleda de sempre (chard landrace), col de tocino (cabbage landrace), or enciam negre (lettuce landrace) are landraces that respondents value because they are kept throughout the winter, they are cold and snow resistant, and so they do not freeze during the winter. However, due to warmer conditions, some landraces are not as valued as before since now they have a shorter harvest period, such as enciam negre (lettuce landrace), which spikes more rapidly than other commercial varieties.Despite this loss of crop diversity, there is still an important maintenance of landraces in home gardens . This maintenance is due to organoleptic properties since home gardeners have a preference for certain tastes and smells associated with landraces that commercial varieties lack. Another important reason for this maintenance is the adaptation of landraces to the local climate, specifically to cold and snow. For example, ceba valenciana (onion landrace), pomera de Cardós (apple tree landrace), and pomera morro de llebre (apple tree landrace).Other reasons are the cultural and traditional appreciation of certain landraces and the maintenance of dishes and culinary preparations that involve these landraces. Also, the diversity of landraces allows some crop species to be present throughout the year; this is the case with cabbage. Home gardeners have different landraces of cabbage that are harvested in different seasons, and in doing so, they can eat cabbage all year round. Similarly, some landraces are valued for their long conservation, such as The last reason that respondents mentioned related to the importance of maintaining landraces is the fact that traditional seeds sometimes give more productive crops than commercially grown and bought plants. This depends on the type of crop species and landrace. For example, two farmers complained that when they buy a potato plant, it is not as productive as when they plant their own landrace. Others also mentioned that when they buy a plant, it already has pests or diseases (such as the potato beetle), so they prefer to sow themselves the landraces and avoid buying the commercial variety.In this section, we discuss our results focusing on (1) the perceived impact of climate changes on home gardens and changes in management practices to adapt to those impacts, (2) the changes in crop species and landraces, and (3) the climatic and socioeconomic factors driving those changes. Before all else, we account for a set of caveats to interpret the results correctly.First, the list of landraces stems from respondents’ individual reports, without the possibility of conducting focus group discussions to get a cultural consensus agreement about landraces due to the COVID-19 sanitary situation. Therefore, there may be a repetition of the same landrace since it could be named differently in different towns or households. Similarly, two different landraces may have the same name, but different genotypes. To solve this limitation, we used a previously published catalogue of landraces of the area and conducted additional interviews to solve incongruences as stated in the methods section.Second, although the classification of the local indicators of climate change impacts has been carried out according to the LICCI protocol . We deeply discuss this systemic interrelation in the subsequent section.As several previous studies have pointed out, the perception of climate change impacts often drives the implementation of adaptation strategies in agricultural systems  can largely reduce the potential impacts of climate change. This is why respondents were asked about changes in management practices in their home gardens  were not grown before because the climate was cooler and there was more humidity than nowadays. That is in line with the physiological characteristics of this crop species since pepper has a temperature range for germination higher than cool-season vegetables and low temperatures can reduce fruit quality . The drivers of this loss are in line with the crop species changes mentioned before, including the increasing use of commercial seeds, the abandonment of cattle farming and rainfed crops, and the increase of crop pests and diseases. The overall diffusion of modern varieties has produced a decrease in the number of landraces and therefore an increase of the genetic erosion in agriculture, as reported in the literature for other parts of the world Negri . LandracEven so, people have expressed a preference for landraces rather than for commercial varieties and that is why they keep maintaining or have increased the cultivation of 28 landraces in home gardens, which is consistent with findings in previous studies in the high-mountain agroecosystems of the Iberian Peninsula, such as in Vall Fosca