This emotion is evoked more strongly when the observer anticipates that other people will be reciprocally cooperative. Coalitionality should therefore moderate feelings of elevation, as whether the observer shares the coalitional affiliation of those observed should influence the observer's assessment of the likelihood that the latter will cooperate with the observer. We examined this thesis in studies contemporaneous with the 2020 Black Lives Matter (BLM) protests. Although BLM protests were predominantly peaceful, they were depicted by conservative media as destructive and antisocial. In two large-scale, pre-registered online studies , political orientation strongly moderated feelings of state elevation elicited by a video of a peaceful BLM protest (Studies 1 and 2) or a peaceful Back the Blue (BtB) counter-protest (Study 2). Political conservatism predicted less elevation following the BLM video and more elevation following the BtB video. Elevation elicited by the BLM video correlated with preferences to defund police, whereas elevation elicited by the BtB video correlated with preferences to increase police funding. These findings extend prior work on elevation into the area of prosocial cooperation in the context of coalitional conflict.Witnessing altruistic behaviour can elicit  Here, we examine the role of partisan attitudes in shaping emotional responses within the context of a deeply moralized, politically divisive, ongoing societal conflict: the Movement for Black Lives in the United States . AlthougThe various experiences commonly described as ‘emotions’ can be understood as the product of corresponding adaptations produced by natural selection. Emotion adaptations are composed of transitory entrainments of diverse perceptual, somatic and cognitive mechanisms that generate responses which, on average, effectively addressed recurring challenges and opportunities in the social and physical lives of ancestral hominids over evolutionary time –4. For eThe Attitude-Scenario-Emotion (ASE) framework models the process by which representations acquired through experience moderate emotional reactions in a circumstantially contingent, fitness-enhancing way . In the moral elevation, an emotion designed to heighten prosociality upon witnessing the exceptionally prosocial behaviour of others  attitudes will correlate with greater [less] elevation following a BLM protest video. (Tested in Studies 1 and 2)b. Liberal [conservative] attitudes will correlate with less [greater] elevation following a BtB protest video. (Tested in Study 2)c. Political orientation will not predict elevation in an apolitical, affectively neutral control video condition. (Tested in Studies 1 and 2)Political orientation will moderate elevation in response to video stimuli depicting each protest movement. 2a. Elevation in the context of a BLM protest video will positively correlate with preferring to decrease police funding. (Tested in Studies 1 and 2)b. Elevation in the context of a BtB protest video will positively correlate with preferring to increase police funding. (Tested in Study 2)c. Elevation will not predict funding preferences in the neutral video condition. (Tested in Studies 1 and 2)Partisan elevation will track partisan police funding preferences.To confirm that our measure of political orientation-tracked attitudes toward the police and the BLM protest movement as intended, we also planned to check whether: –Liberal [conservative] political attitudes track preferences to decrease [increase] police funding. –Political attitudes track appraisals of the BLM movement, BtB movement and police as prospective prosocial cooperators.The ASE framework, articulated with models of partisan cooperation under intergroup conflict and applied to the BLM movement, generates a constellation of related predictions:. 6. 6.1https://osf.io/kdeg6/). All studies were approved by the University of California, Los Angeles, Institutional Review Board and performed in accordance with guidelines governing research with human participants. Informed consent was obtained from all participants prior to beginning each study.The pre-registrations, full materials and datasets for the studies reported here are publicly archived . 71.4% of the participants identified as White, 11.5% as Black, and 17.1% as Other. With respect to political party affiliation, 27.8% identified as Republicans, 47.5% identified as Democrats and 24.8% identified as Independents.Based on results from our prior work , as well. 6.1.2α = 0.95; for descriptives, see electronic supplementary material, table S1).In order to confirm that political conservatism indexes attitudes viewing police as prosocial cooperative partners, participants first completed a face-valid self-report measure consisting of five items assessing perceptions of the degree of prosociality characteristic of police . The BLM video was composited from several contemporaneous media reports, edited with the intent of highlighting (i) the general aim of the protesters to increase racial equity in policing, (ii) prosocial coordination among the protesters and (iii) expressions of overt anger regarding police violence. A melodic musical score was layered over this composite of footage to render it more cohesive and to cue participants that the video portrays the BLM movement in a positive light. In sum, the BLM video was intended to elicit state elevation among politically liberal participants.α = 0.97) previously developed by Sparks et al.  would indicate uniformly conservative [liberal] positions. We used political orientation as a measure of coalitional left- versus right-wing attitudes toward the BLM movement. Once the study was complete, participants were thanked, debriefed and compensated.  which are intended for separate publication.)Finally, we measured political orientation as part of the demographic questions, using a modification of Dodd et al.'s  version et al.'s  issues i. 7. 7.1r(854) = 0.62, p < 0.001, and with idealistic attitudes toward the police as trustworthy and prosocial, r(854) = 0.49, p < 0.001 . In sum, political orientation does appear to capture overall perceptions of the BLM movement. . Follow-up tests did not reveal an interaction between political orientation and video condition on funding preference, p = 0.668.As anticipated, and in support of the use of overall political orientation as an index of attitudes toward the BLM movement, political conservatism positively correlated both with preferences to increase police funding, . 7.2M = −0.23, s.d. = 0.40. Consistent with this left-leaning tendency, an analysis of variance (ANOVA) revealed state elevation to be greater in the BLM video condition, M = 1.52, s.d. = 0.91, than in the Control condition, M = 0.62, s.d. = 0.91 , b = −1.09, s.e. = 0.13, t = −8.25, p < 0.001. Simple slopes analyses confirmed that, in line with Prediction 1, conservative attitudes were negatively associated with elevation in the BLM condition, b = −0.82, s.e. = 0.09, t = −8.69, p < 0.001, 95% CI = , but not the Control condition .The political orientation of the overall sample was left-leaning, ondition . Against. 7.3M = −7.07, s.d. = 48.21, to a greater extent than in the Control condition, M = −1.11, s.d. = 48.52 . We next tested the association between state elevation elicited by the BLM video manipulation and preferences to decrease versus increase funding for the police. A moderated linear regression model including police funding preferences as the dependent variable, state elevation as the independent variable, and video condition as the potential moderator, revealed a significant interaction, b = −28.48, s.e. = 4.08, t = −6.99, p < 0.001. Consistent with Prediction 2, simple slopes analyses confirmed that state elevation in the BLM video condition-tracked preferences to defund the police, b = −14.40, s.e. = 2.49, t = −5.77, p < 0.001, 95% CI =  An ANOVA revealed a nonsignificant trend for participants to favour decreasing police funding in the BLM video condition, , −9.48] . In the . 8In Study 1, we assessed the role of political orientation in moderating feelings of elevation elicited by a pro-BLM video relative to a neutral control video. As predicted, liberal [conservative] attitudes predicted significantly heightened [diminished] feelings of elevation in response to the BLM video relative to the control video, and feelings of state elevation engendered by the BLM video-tracked preferences to defund the police. This overall pattern supports the premise that partisan coalitional attitudes can moderate feelings of state elevation in response to cues of cooperation. However, the design of Study 1 failed to assess effects of cues of cooperation likely to elicit elevation among politically conservative individuals. Accordingly, in Study 2, we added a BtB video manipulation closely patterned after the BLM video created for Study 1.Although the BLM video increased state elevation, there was no significant effect of the video condition, nor interaction between political orientation and video condition, on police funding preferences. These findings suggest that funding preferences reflected relatively stable political attitudes correlated with the propensity to experience coalitional elevation, rather than a decision regarding whether to behave prosocially that would theoretically be susceptible to the influence of state elevation.Unexpectedly, political conservatism was modestly yet significantly positively correlated with state elevation in the control video condition. Speculatively, given the politicized nature of the COVID-19 pandemic in the USA  and the . 9. 9.1. 9.1.1ageM = 41.9 years, s.d. = 13.1). Of the participants, 73.2% identified as White, 9.0% as Black and 17.8% as Other; 24.0% identified as Republicans, 49.1% identified as Democrats and 26.9% identified as Independents.We targeted a sample size of 1500 (500 per condition), recruited via Amazon Mechanical Turk  in exchange for $1.25. Participants were recruited on 18 December 2020. Data were prescreened as in Study 1, yielding a final sample consisting of 1316 participants , as well as a parallel measure added in Study 2 assessing perceptions of the BLM protesters (α = 0.94) . Next, in a between-subjects design, participants were randomly assigned to view a video depicting either BLM protests, BtB protests or a neutral control . The BLM video was the same as used in Study 1. Paralleling the BLM video, the BtB video was composited from contemporaneous media reports, edited with the intent of highlighting (i) the general aim of the counter-protesters to refute the criticisms of the BLM protesters and support existing police institutions, (ii) prosocial coordination among the protesters and (iii) expressions of overt anger regarding the BLM protests. The same musical score was layered over the BtB footage as used in the BLM video, and the BtB sequence was patterned closely after the BLM video (see OSF). In sum, just as the BLM video was intended to elicit state elevation in politically liberal participants, the BtB video was intended to elicit state elevation in politically conservative participants.As in Study 1, participants completed a self-report measure assessing perceptions of the degree of prosociality characteristic of police  .As in Study 1, the political orientation of the overall sample was somewhat left-leaning, mparison  revealedps < 0.001, in the associations between political orientation and elevation when contrasting the BLM and BtB conditions, b = −2.18, s.e. = 0.12, t = −17.62, the BLM and Control conditions, b = −1.17, s.e. = 0.12, t = −9.53, and the BtB and Control conditions, b = 1.02, s.e. = 0.12, t = 8.23. Consistent with Prediction 1, simple slopes analyses confirmed that conservative political attitudes were negatively associated with elevation in the BLM video condition, b = −0.89, s.e. = 0.09, t = −10.20, p < 0.001, 95% CI = , but positively correlated with elevation in the BtB video condition, b = 1.30, s.e. = 0.09, t = 14.70, p < 0.001, 95% CI =  A moderated linear regression model including state elevation as the dependent variable, political orientation as the independent variable and video condition as the potential moderator revealed significant interactions, 2, 1.47] . Against. 10.3F2,1312 = 3.88, p = 0.021). Pairwise comparisons revealed that participants favoured decreasing the percentage of current police funding in the BLM video condition, M = −4.31, s.d. = 44.59, to a greater extent than in the Control condition, M = 2.93, s.d. = 44.75, p = 0.044, 95% CI = , comparably to the trend observed in Study 1. There was also a significant difference between police funding preferences in the BLM video condition relative to the BtB condition , p = 0.040, 95% CI = , with no difference in police funding preferences between the Control condition and the BtB video condition, p = 0.998 .An ANOVA found differences across conditions in funding preferences We next tested the association between state elevation elicited by the video manipulation and police funding preferences. A moderated linear regression model including police funding preferences as the dependent variable, state elevation as the independent variable and video condition as the potential moderator revealed significant interactions, , 34.30] . In the . 11In Study 2, we closely replicated our prior findings that political attitudes moderate feelings of elevation in response to the BLM video, and that state elevation elicited by the BLM video tracks preferences to defund the police. Further bolstering the hypothesis that support versus opposition to the BLM movement would determine state elevation, conservatism predicted greater elevation in response to the BtB video and elevation aroused by the BtB video correlated with preferences to increase police funding. Unexpectedly, despite the use of an alternate video intended to avoid political or affective connotations, as in Study 1, conservatism also mildly positively correlated with state elevation in the control condition, arguably making the inverse correlation in the BLM condition somewhat more striking. However, given the notably low levels of elevation in the control condition, this pattern merely suggests that conservatives are disposed to experience a slight degree of positive affect when viewing individuals engaged in productive tasks (e.g. repairing a broken computer).p = 0.044) despite use of a relatively large sample, (ii) the contrast was non-significant in the previous study and (iii) there was no comparable funding preference shift in the BtB condition. Thus, although there do appear to be indications in both studies that exposure to the BLM video may have mildly inclined left-leaning participants to favour the movement's call to decrease police funding, funding preference ratings appear to have primarily reflected stable coalitional attitudes rather than outputs of state elevation.In a departure from the trend observed in Study 1, police funding preferences were significantly lower in the BLM video condition relative to the control condition. However, this finding should be treated with caution, as (i) the significance level was marginal , albeit primarily or exclusively directed toward the in-group. An important but unanswered question is whether elevation would similarly motivate altruistic acts of destruction or violence on behalf of one's coalition. On the one hand, conceptually, prosociality encompasses righteous punishment of those perceived to be wrongdoers or threats  and may entiment ,66. WithWe observed striking interactions between coalitional attitudes and state elevation in response to stimuli related to a predominantly peaceful political conflict. Given the ubiquity of group conflicts of varying degrees of intensity over the course of human evolution, coalitional sentiments should be sensitive to the relative fitness stakes of conflict, suggesting that the interactions between group attitudes and state elevation observed in the present studies will be more pronounced in contexts of overtly violent intergroup conflict. For example, at the time of writing, the Russian Federation has invaded a sovereign nation, Ukraine, under a flimsy pretext of preemptive self-defense. Among members of nations antagonistic to Russia such as the United States, this has elicited outrage, donations of humanitarian aid and praise of Ukrainian acts of military resistance as heroically altruistic; indeed, a legion of foreign volunteer fighters is serving on the front lines . The waret al. [Whereas our experiments probed the effects of witnessing cooperative protest behaviour dedicated to coalitional ends, the extent to which similar coalitional dynamics apply when observers witness non-coalitional acts of overt prosociality performed by individuals displaying markers of coalitional identity (e.g. a ‘Black Lives Matter’ or ‘Make America Great Again’ t-shirt) remains unclear. To the extent that an altruist's membership in one's own ingroup or an antagonistic out-group indexes the probability of cooperation beneficial to the observer, such markers of coalitional affiliation can be expected to moderate the capacity of their prosocial acts to elicit elevation. Further research might test whether coalitional affiliation comparably determines elevation (and related helping tendencies) even when the observed prosocial acts are unrelated to group conflict. Relatedly, for instance, Blomster Lyshol et al.  found thet al. [et al. [et al.'s participants been assigned a video depicting large-scale, peaceful coordination among Palestinians demanding just treatment akin to the BLM video used here, the Israeli sample may not have evinced increased support for humanitarian policies toward Palestinians—and might instead have favoured less humane policies—to the extent that their baseline attitudes guided interpretation of the protest stimulus as coalitionally antagonistic. Although this prediction requires empirical testing, a translational lesson would obtain if confirmed: to enhance intergroup sympathy, eschew coalitional framing.Other studies assessing the impact of elevation-elicitation on group relations have used prosocial primes that are incidental to coalitional identification or active conflict. For example, Oliver et al.  found thet al. ). Althou [et al.  recentlyOur functionalist account of elevation as a mechanism that shapes costly behaviour so as to maximize payoffs for the actor predicts that, in coalitional contexts, elevation-elicitation will hinge on the personal relevance of the coalitions with respect to the likely benefits or risks of cooperating. Emerging work indicates that, even in quite small communities, there is substantial variation in attitudes concerning, and willingness to be prosocial toward, out-groups—variation that is largely explicable in terms of the positive and negative affordances of out-groups for each individual . Crucialet al. [Freeman et al.  found thet al. ), such tkama muta, the feeling of being ‘moved’ or ‘touched’ associated with the sudden intensification of a communal sharing relationship, that is, one in which the parties are equivalent in some key aspect of identity [Elevation is similar to—perhaps even isomorphic with—a hypothesized emotion that researchers have labelled identity . Kama muidentity ,78. Analidentity  finds frThe present findings highlight both the promise of elevation to inspire cooperation in pursuit of political aims and the hazard of elevation as potentially escalating coalitional conflict in ways which exacerbate harm to all sides. Research indicates that upholding highly moralized coalitional objectives  is viewed as an imperative duty, and therefore not subject to compromise, regardless of utilitarian cost-benefit considerations . Elevati. 13In both scholarly literature and popular accounts, elevation and other positive emotions are sometimes presented as if they were a panacea for healing social divisions—as though human nature were designed such that mere exposure to selfless deeds would necessarily evoke recognition of our shared affinity. However, from a functional perspective, emotion adaptations such as elevation evolved to promote individual fitness, and relative fitness payoffs predict when and how emotions are elicited. In situations of intergroup conflict, prosocial behaviour in the service of coalitional goals evokes elevation in observers who side with those prosocial actors, but not in those who align with the opposing side. Actions that inspire elevation and altruistic self-sacrifice on the part of some observers will be met with cold indifference, or even hostility, contingent on representations of the affordances of individuals or groups vis-à-vis the observer's welfare.


Intra-uterine growth restriction (IUGR) is a common cause of fetal/neonatal morbidity and mortality and is associated with increased offspring predisposition for cardiovascular disease (CVD) development. Mitochondria are essential organelles in maintaining cardiac function, and thus, fetal cardiac mitochondria could be responsive to the IUGR environment. In this study, we investigated whether in utero fetal cardiac mitochondrial programming can be detectable in an early stage of IUGR pregnancy. Using a well-established nonhuman IUGR primate model, we induced IUGR by reducing by 30% the maternal diet (MNR), both in males (MNR-M) and in female (MNR-F) fetuses. Fetal cardiac left ventricle (LV) tissue and blood were collected at 90 days of gestation . Blood biochemical parameters were determined and heart LV mitochondrial biology assessed. MNR fetus biochemical blood parameters confirm an early fetal response to MNR. In addition, we show that in utero cardiac mitochondrial MNR adaptations are already detectable at this early stage, in a sex-divergent way. MNR induced alterations in the cardiac gene expression of oxidative phosphorylation (OXPHOS) subunits , along with increased protein content for complex-I, -III, and -IV subunits only for MNR-M in comparison with male controls, highlight the fetal cardiac sex-divergent response to MNR. At this fetal stage, no major alterations were detected in mitochondrial DNA copy number nor markers for oxidative stress. This study shows that in 90-day nonhuman primate fetuses, a 30% decrease in maternal nutrition generated early in utero adaptations in fetal blood biochemical parameters and sex-specific alterations in cardiac left ventricle gene and protein expression profiles, affecting predominantly OXPHOS subunits. Since the OXPHOS system is determinant for energy production in mitochondria, our findings suggest that these early IUGR-induced mitochondrial adaptations play a role in offspring’s mitochondrial dysfunction and can increase predisposition to CVD in a sex-specific way. Cardiovascular disease (CVD) incidence is increasing worldwide at an alarming rate, especially among the young adult population ,2. In coIt is now well accepted that maternal nutrition influences the intrauterine environment and, consequently, the offspring’s short- and long-term health ,5. MaterUsing a well-established nonhuman IUGR primate model of moderate maternal nutrient reduction (MNR), which consisted of a 30% nutrient reduction in comparison with control mothers, and that shares a high level of gene homology with humans (94%) , we prevDespite the glycolytic dependence of fetal cardiomyocytes  and thatPapio spp.) with similar morphometrics, including pre-pregnancy body weight (13.9 ± 0.5 vs. 14.26 ± 0.6), were randomly assigned to the Control (C) or MNR group. After diet treatment, maternal weight variation differed between groups, being increased for MNR mothers vs. C mothers   .p = 0.047). For the same group, C-F fetal body mass index was decreased vs. C-M . Fetal blood biochemical analysis revealed that fetal glucose circulating levels were decreased for MNR-F vs. C-F . This was not observed for maternal blood glucose concentration. Interestingly, fetal blood urea nitrogen (BUN) was decreased for MNR vs. C , being more evident for MNR-F vs. C-F , and for C-F vs. C-M . Fetal BUN/creatinine ratio was decreased for MNR vs. C   .The cluster heatmap analysis identified a reasonable separation between the experimental groups’ amino acid blood levels, resulting in well-formed clusters as seen in the heatmap . In geneND1 versus the absolute amount of the B2M nuclear gene for each sample. No statistically significant differences were detected for LV cardiac mtDNA copy number at 0.5 G between C and MNR offspring , BCS1L  were downregulated, whereas the genes NDUFB6 , NDUFB3 , UQCRC2 , ATP5O , and ATP5A1  were upregulated. Moreover, two genes had a tendency to be downregulated ), and five genes had a tendency to be upregulated (COX6C (p = 0.0714), BAK1 (p = 0.0856), ATP5J (p = 0.0588), TIMM10 (p = 0.0762), ATP5F1 (p = 0.0595)).Human Mitochondrial Energy Metabolism and the Human Mitochondria Pathway Arrays were used to evaluate MNR-induced in utero fetal cardiac RNA transcriptional changes at 0.5 G. The expression levels of all of the evaluated genes are summarized in the heatmap and the scatter plot in ATP5A1 was upregulated and LHPP was downregulated in both MNR-M  and MNR-F  in comparison with the control of the respective sex. For MNR-M , NDUFV3 , NDUFB10 , and COX10 was tendentially upregulated (p = 0.0827). For the same group, along with LHPP, the transcripts for UCP2 , SLC25A31 , IMMP1L , TIMM50 , and TIMM22  were downregulated in comparison with C-M, and another seven transcripts were tendentially downregulated, SLC25A2 (p = 0.0677), PMAIP1 (p = 0.0770), CPT2 (p = 0.0958), TP53 (p = 0.0577), SOD2 (p = 0.0620), BID (p = 0.0759), and BBC3 (p = 0.0848).Gene expression variations are sex-specific . In commor MNR-M , along wATP5A1 was upregulated for MNR-F: NDUFB3 . For the same group, 10 genes were downregulated along with LHPP: HSPD1 , CYC1 , UQCRC1 , SDHB , NDUFA10 , COX4I2 , NDUFS2 , BCS1L , ATP4A , along with other four tendentially downregulated transcripts, COX7A2L (p = 0.0671), NDUFA3 (p = 0.0942), and two tendentially upregulated, SLC25A5 (p = 0.0868), NDUFB6 (p = 0.0974).In comparison with C-F , only 1 ATP5A1, ATP5G3, HSPD90AA1, IMMP1L, SLC25A2, SLC25A31, TIMM22, TOMM70A, and UCP2, with ATP5C1, COX6A2, NDUFV1, CPT2, DNM1L, MFN1, OPA1, and TP53, being tendentially upregulated, and for MNR-F, the upregulated genes were COX4I2, NDUFV3, CYC1, MIPEP, TOMM40, ATP4A, UQCRC1, UQCRFS1, SDHB, SDHD, NDUFA11, NDUFC1, NDUFS2, NDUFS3, TOMM20, BCS1L, NDUFA1, NDUFA10, COX412 with ATP5B, LHPP, HSPD1, SLC25A19, TIMM17A, TIMM44, and TOMM22, being tendentially upregulated.When comparing the fold-regulation of gene expression in MNR-M vs. MNR-F, NDUFB10, NDUFA3, NDUFV3, NDUFS2, SDHB, UQCRC1, COX7A2L, COX4I2, and CYC1 for MNR-F vs. C-F; these transcripts tended to be downregulated. On the contrary, the group of transcripts that tended to be downregulated for MNR-M vs. C-M was as follows: BBC3, BID, CPT2, PMAIP1, SLC25A2, SLC25A31, SOD2, TIMM22, TIMM50, and TP53; these were upregulated for MNR-F vs. C-F. Nevertheless, a few genes show similar expression behaviors for both sexes .Overall, fold-regulation patterns are patented in p = 0.0485), complex III UQCRC2 , and complex IV COXII  subunits, in which OXPHOS protein content overall increased for MNR-M vs. C-M. Additionally, in MNR-F fetuses there was an increase in LV protein content for the complex IV COX6C subunit protein  . Protein indicators of mitochondrial mass, dynamics, oxidative stress, and OXPHOS subunit proteins were semi-quantified . Protein 0.0063) .p = 0.0285), complex III UQCRC2 , complex IV COXII , and ATP synthase ATP5A1 subunit . Mitochondrial biogenesis and mass regulator mitochondrial transcription factor A (TFAM) (p = 0.05), as well as the translocase of the outer mitochondrial membrane 20 (TOM20) (p = 0.0004), respectively, were also increased for C-F vs. C-M. Comparing both MNR groups, the sexual dimorphism regarding mitochondrial protein amount was attenuated with only OXPHOS complex IV COX6C subunit  and TOM20  being increased for MNR-F vs. MNR-M.Cardiac mitochondrial sexual dimorphism was well evidenced in the protein levels between the control groups  and proteins involved in macromolecular assembly (HSPD1 and BCS1L). It is worth mentioning that BCS1L plays an important role in MRC-complex III assembly [BCS1L and OXPHOS impairment has been proposed as a mechanistic link in prostate cancer-related fatigue development; however, more studies are needed to fully understand if this link between BCS1L gene expression and complexes activities is verified for other conditions. Nevertheless, transcripts for UQCRC2, a gene encoding for complex-III subunit, was upregulated for MNR fetuses, along with other OXPHOS complex subunits: complex-I , -IV (COX6C), and ATP synthase . This represents more than half of the measured genes whose expression was altered by MNR in the cardiac LV. In addition, and highlighting MNR-induced mitochondrial alterations, the protein amount of citrate synthase, a mitochondrial matrix enzyme commonly used as a mitochondrial marker [NDUFB6, and ATP synthase ATP5A1 were upregulated. These transcript alterations did not result in many protein content alterations for the same subunits, in both fetal time points (with the exception of COX6C for MNR mid-gestation fetuses), possibly due to the dynamic processes between the genome and the protein levels, which include transcription, splicing, and translation [SLC25A2), mitochondrial ADP/ATP exchange (SLC25A31), uncoupling of mitochondrial oxidative phosphorylation (UCP2), molecular folding (HSP90AA1), removal of the mitochondrial targeting pre-sequence of nuclear-encoded proteins (IMMP1L), and protein translocation . Interestingly, both complex III-core 2 subunit gene expression (UQCRC2) and protein content were increased for MNR-M vs. C-M. Moreover, complex-I (NDUFB8) and -IV (COXII) subunit protein contents were increased for MNR-M vs. C-M, whereas no differences were detected in MNR-F. Thus, MNR seems to impact more severely male fetuses’ OXPHOS protein content, whereas for female offspring, these alterations may become more pronounced at a later developmental stage or even at a postnatal stage, since we did not detect differences in these subunits’ protein content for term female fetuses in our previous study [Cardiac gene expression analysis of mitochondria-related genes revealed that MNR-induced gene downregulation involves genes that encode for a phosphatase enzyme  approved all animal procedures, including pain relief. These were conducted in the Association for Assessment and Accreditation of Laboratory Animal Care-approved facilities and NIH Guide for the Care and Use of Laboratory Animals.Papio spp.) of a similar morphometric phenotype were selected for the study. Animals were housed at the Southwest National Primate Research Center at the Texas Biomedical Research Institute (TBRI) in the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-approved facilities. Housing conditions and animal caging were performed as previously described [As previously described , maternaescribed .Each baboon’s weight was obtained while crossing an electronic scale , and as described elsewhere .Normally cycling female baboons from 8 to 15 years old were observed twice a day for well-being and three times a week for turgescence  and signs of vaginal bleeding to assess their reproductive cycle and enable determining the timing of pregnancy . After aPurina Monkey Diet 5038, standard biscuits were provided once a day. The biscuit is described as a “complete life-cycle diet for all Old-World Primates” and contains stabilized vitamin C as well as all other required vitamins. The basic composition includes crude protein (≥15%), crude fat (≥5%), crude fiber (≤6%), ash (≤5%), and added minerals (≤3%) .After the confirmation of pregnancy, food intake was recorded in 8 female baboons fed ad libitum and was calculated as 50.61 ± 3.61 kcal/kg of body weight per day. Before the start of the controlled diet, baboons were fed the same diet without a biscuit limit. Water was continuously available in the feeding cages via individual waterers  and at several locations in the group housing. Animal food consumption, weights, and health status were recorded daily. More details regarding housing and environmental enrichment have been previously published .Mothers were fasted from their last feeding time the day before, until the cesarean section . A fullyND1  and for B2M  at 500 nM each. The amplification of 25 ng DNA was performed with an initial cycle of 2 min at 98 °C, followed by 40 cycles of 5 s at 98 °C plus 5 s at 60 °C. At the end of each cycle, Eva Green fluorescence was recorded to allow Ct determination. For quality control, the melting temperature of the PCR products was determined after amplification by performing melting curves, and no template controls were run.DNA extraction was performed as previously described . RT-PCR 9 copies/μL, and tenfold serial dilutions were prepared. mtDNA copy number was determined by the ratio between the absolute amounts of mitochondrial gene ND1 versus the absolute amount of the B2M nuclear gene in each sample, using the CFX96 Manager software .For absolute quantification and amplification efficiency, standards at known copy numbers were produced by purifying PCR products. After optimizing the annealing temperature, products were amplified for each primer pair using the HotstarTaq Master Mix Kit . Briefly, 1 μL of a DNA sample was added to a PCR tube containing the HotStar Taq Master Mix and the specific primers and placed in a CFX96 real-time PCR system. The amplification protocol started with an initial activation step of 15 min at 95 °C degrees, followed by 35 cycles of 1 min at 94 °C (denaturation) plus 1 min at 60 °C , plus 1 min at 72 °C (extension), and a final extension step of 10 min at 72 °C. After amplification, the products were purified using the MiniElute PCR purification kit (#280006 Qiagen) following the manufacturer’s instructions. Eluted DNA was quantified in a Nanodrop 2000 device, the copy numbers were adjusted to 5 × 102 Profiler polymerase chain reaction (PCR) Array System , SA Biosciences , Qiagen ), was used to evaluate the different cardiac mitochondrial transcripts between control and MNR fetuses as previously described [HPRT1), ribosomal protein L13a (RPL13A), and Beta-actin (ACTB) and analyzed with the ΔΔCt method (where Ct is threshold cycle) using the PCR Array Data Analysis Web Portal (SA Biosciences). The transcripts used in this study are listed in RNA extraction was performed following the protocol previously described by Cox et al. . RNA wasescribed . Each PCg for 5 min. Equivalent amounts of total protein (10 μg per lane) were loaded in a 10–20% gradient Tris-HCl polyacrylamide gel as well as two different standards for molecular weight estimation and for monitoring electrophoresis progress, the Precision Plus Protein Dual Color Standards (Bio-Rad) and the SeeBlue Plus2 Pre-Stained Standard , Invitrogen ). Electrophoresis was carried out at room temperature in a Criterion system (Bio-Rad) using 150 V until the sample buffer (blue) reaches the bottom of the gel (≈90 min). After separation by SDS-PAGE, proteins were electrophoretically transferred in a TransBlot Cell system (Bio-Rad) to a polyvinylidene difluoride (PVDF) membrane previously activated, a constant amperage (0.5 A) for 2 h at 4 °C using a CAPS transfer buffer (10 mM 3-(Cyclohexylamino)-1-propanesulfonic acid pH 11 (NaOH), 10% methanol). The quality of the electrophoretic transfer was evaluated by the complete transfer of pre-stained molecular weight markers below 100 kDa and via Ponceau staining. Ponceau results were also used to confirm an equal amount of protein loading and to normalize band density. After Ponceau removal, the membranes were blocked in 5% non-fat milk/PBS overnight at 4 °C with agitation. Before incubation with primary antibodies, the membrane was washed for 10 min in PBS 0.05% Tween-20 (PBS-T). Primary antibodies were prepared in 1% non-fat milk/PBS to a final volume of 5 mL and incubated overnight at 4 °C. After incubation with primary antibodies, membranes were washed with PBS-T solution three times, 5 min each, and incubated with the correspondent alkaline phosphatase-conjugated secondary antibodies for 2 h at room temperature with stirring. For immunodetection, membranes were washed three times for 5 min each with PBS-T, rinsed in PBS to remove any Tween-20, which can be inhibitory to the detection method, dried, and incubated with an enhanced chemifluorescence (ECF) system  during a maximum of 5 min. Density analysis of bands was carried out with VisionWorks LS Image Acquisition and Analysis Software (UVP). The resulting images were analyzed and densities were normalized to Ponceau. The average value of the C-Males (M) group was assumed as one unit, and the values of each sample were determined proportionally. All of the primary antibodies used in this experiment were purchased from abcam: NDUFB8 , UQCRC2 , MT-CO2 , COX6C , ATP5A , TOMM20 , TFAM , and citrate synthase . All of the secondary antibodies were purchased from Santa Cruz and were used in a 1:5000 dilution: rabbit (sc-2007), mouse (sc-2008), goat (sc-2771).Protein analyses via Western blotting were performed following standard protocols . Sample The software GraphPad Prism version 8.0  was used for data analysis. Each pregnant baboon and the corresponding fetus were considered an experimental unit. Outbred pregnant female baboons were randomly assigned to control or MNR groups. Data are expressed as mean or as mean ± SD. Normality was assessed via the Kolmogorov–Smirnov or Shapiro–Wilk tests. To assess the effect of MNR on fetal cardiac mitochondrial parameters, the following comparisons were made: (1) only to evaluate diet-induced alterations, independently of fetal sex: between Control (C) vs. MNR; (2) to simultaneously assess the impact of diet and sex: between male control (C-M) vs. female control (C-F), male MNR (MNR-M) vs. C-M, female MNR (MNR-F) vs. C-F, and MNR-M vs. MNR-F. The orange software (version 3.32.0) was used for the computational data analysis and visualization. Clustering (opt ordering) was applied to both columns and rows. Data were normalized for value ranges between −1 and 1.In this study, 30% MNR led to sex-specific alterations on the NHP fetal cardiac left-ventricle mitochondrial-related gene expression at 90 days old (0.5 G), some of which persisted until near term (0.9 G). More than half of the MNR-induced gene expression alterations encode for OXPHOS complex subunits. Given that the OXPHOS system is primarily responsible for energy production in the mitochondria, early IUGR-induced mitochondrial adaptations could play a role in IUGR offspring’s increased predisposition to CVD. The fact that we here showed that MNR can induce fetal cardiac mitochondrial function alterations as early as the mid-gestation period highlights the need to better understand the early origins of CVD, which can provide new targets for disease prevention and mitigation.


This retrospective, single-center, practice-based cohort study aimed to analyze factors associated with the success of removable partial dentures retained by telescopic crowns (TRPD).TRPD which were placed in a single practice of a practice-based research network were analyzed. Data from 139 patients (age (SD): 66 (11) years; 66 female) with 174 TRPD including 488 non-precious alloy telescopic crowns (TC) between 2004 and 2016 were included. TC without any technical complication were considered as successful, and as survived, if they were still in function at the last check-up. Multilevel Cox proportional hazard models were used to evaluate the association between clinical factors and time until failure.5years,TC-level: 5.0%) as well as 136 (87%) TRPD  (“worst-case scenario”) and 150 (86%) TRPD  (“best-case scenario”) were considered as successful. The main failure types were recementation (n = 39), endodontic treatment (n = 36), and extraction (n = 35). TC in male patients showed 1.6 times higher risk for failure than in female patients . TC on premolars showed 2.2 times higher risk for failure than on incisors  and TC in dentures with ≤ 3TC showed 2.1 times higher risk for failure than TC in dentures with > 3TC . Furthermore, TC on the most distal tooth in an arch showed 2.4 times higher risk for failure than TC on a more mesial tooth .Within a mean follow-up period (SD) of 4.2 (3.3) years (min–max: 1 day–12 years), 372 (76%) TC (AFRFor removable partial dentures retained by telescopic crowns, high success rates could be found after up to 12 years. Patient-level and tooth-level factors were significantly associated with failure.For removable partial dentures retained by telescopic crowns, high success rates could be found after up to 12 years. Patient-level and tooth-level factors were significantly associated with failure. In an aging world population, dental care and the knowledge regarding prevention of caries are improving, resulting in more retained teeth than past generations had . FurtherTelescopic crowns (TC) represent a specific double crown type with parallel or nearly parallel walled (0–2°) primary crowns. Primary or inner crowns are cemented on the abutment teeth while the secondary or outer crowns are incorporated within the TRPD. Due to the parallelism of the contact surfaces of the primary and secondary crowns, firstly, retention force is created to keep the TRPDs in position  and, secDepending on the different types of double crowns, cumulative survival rates differed between 79 and 95% for frictional telescopic systems after 5–7 years , 12, 13,Thus, the aim of the present retrospective, single-center, practice-based cohort study was, firstly, to evaluate the longevity of different removable partial dentures retained by non-precious alloy telescopic crowns and, secondly, to analyze factors influencing the success of these dentures after up to 12 years.This study was a retrospective, non-interventional, practice-based, clinical study. Reporting follows STROBE guideline for cohort studies. The study has been registered in the German Clinical Trials Register (DRKS-ID: DRKS00026937). Assessment of the status of the restorations was done when patients attended for routine care or recall. According to guidelines for good clinical practice  , the EurPatient receiving removable partial dentures (RPD) solely retained by non-precious alloy telescopic crowns with friction fit (TRPD)Dentists participating in a German dental practice-based research network (Arbeitskreis Zahnärztliche Therapie) were asked to participate. Two dentists in one private practice agreed to search their anonymized electronic patients’ files for the presence of TC. Inclusion criteria were as follows:Patient receiving RPDs with additional retention elements other than TCPatient receiving implant-borne dentures retained by telescopic crowns (TRPD).Exclusion criteria were as follows:The following data were collected anonymously (without reference to patient names) from the electronic patient files of one  practice management program  :On practice-level; dentist;on patient-level; age, sex, date of the first insertion of the TC, date of any (second) restorative (re-)intervention, date of the last visit, number of teeth/restorations (per patient/per the jaw in which the TRPD was worn) being included in the study, number of the regular check-ups and overall visits , insurance status (private or statutory), DMFT.on patient’s tooth-level; type of denture support , number of abutment teeth (≤ 3 vs. > 3), characteristics of the involved tooth , mode of failure, type of restoration.The observation period started with the TRPD being placed. The status of the TRPD was assessed by clinical  and intraoral radiographic examination. The interval for the radiographic examination was defined on an individual basis. Assessment of the TRPD was done in the same practice by the dentist who prepared the TRPD when patients attended for routine care, recall, or when a problem occurred.TC were provided without any technical complication  until the last follow-up examination. Additionally, no abutment tooth loss and no repairable defects occurred, such as abutment tooth fracture, which was, e.g., repairable with post and core build-up. No adaptations of the denture were necessary and the denture stability was not rated as insufficient .Whenever a technical complication was observed or scheduled for one of these treatments at the last check-up — for which an appointment was then made with the patient — the intervention was considered as failed (primary end point: no success).On TRPD-level, two different scenarios were included. Firstly, the failure of the first telescopic crown of a TRPD was considered as failure of the whole TRPD (“worst-case scenario”). Consequently, when all TC of a TRPD were defined as success, the TRPD was also defined as success. Expected interventions such as relining of the denture base or removal of sore spots were not classified as failures. Secondly, the failure of the last telescopic crown of a TRPD was considered as failure of the whole TRPD (“best-case scenario”). Consequently, when all TC of a TRPD were defined as failure, the TRPD was also defined as failure.n = 39) or endodontic treatment (n = 36) was not considered as censored or failed. Contrastingly, the treatment was rated as failure  whenever a TC was renewed or the tooth was extracted ) per TRPD per patient was 2.8 (1.6) (range: 1–9). The mean observation time was 4.2 (3.3) years (maximum: 12 years) Characteristics of teeth are shown in Table 5years) for all dentists and restorations was 5.0% and 1.6%, respectively. The main failure types were recementation of the TC (n = 39), endodontic treatment (n = 36), and extraction of the tooth (n = 35). The success curves of restorations according to the tooth type are shown in Fig. Seventy-six percent of the telescopic crowns (372 out of 488) were considered as successful Table  and 89% 5years for all dentists and dentures were 5.1% and 3.4% respectively.On TRPD-level, 78% (136 out of 174) .Crude bivariate associations between the different baseline characteristics and an increased failure rate are given in Table p = 0.023) higher risk for failure than telescopic crowns in female patients. Furthermore, telescopic crowns on premolars showed 2.2 times (HR: 1.908 (1.1–5.0); p = 0.015) lower risk for failure than on incisors. Telescopic crowns in dentures with ≤ 3 abutment teeth showed 2.1 times (HR: 2.075 (1.3–3.4); p = 0.004) lower risk for failure than telescopic crowns in dentures with > 3 abutment teeth. The position of the TC in the arch was a significant predictor as well. Telescopic crowns on the most distal tooth in an arch showed 2.4 times (HR: 2.429 (1.5–3.8); p < 0.001) higher risk for failure than telescopic crowns on a more mesial tooth.The results of the non-clustered multivariate models including factors possibly associated with an increased failure rate in the bivariate models are shown in Table Predictors being significant for success were also significant for survival  and a median success times of, e.g., the upper (102.5 months) and lower (100.4 months) jaws. Furthermore, only two previous university-based studies on the longevity of TRPD included more dentures than the present study [Due to the retrospective design, no prospective power analysis was feasible in the present study. This is one major limitation, since the retrospective power analysis revealed that the present study may be underpowered to detect moderate to clinically significant relative risks. Consequently, it might be speculated that with a larger sample size or with more failures, the influence of some factors as (significant) predictor and the reliability of the present results would increase . Howevernt study , 13, whint study , 14, 15.Within the limitation of this study, moderate failure rates for removable partial dentures retained by telescopic crowns could be found in a private practice environment after up to 12 years. Patient- and tooth-level factors were significantly associated with failure. Telescopic crowns in male patients, telescopic crowns on premolars, and telescopic crowns in dentures with 4 or more telescopic crowns were significant predictors for failure. However, for telescopic crowns, further studies are needed to improve our knowledge about several of these factors and to analyze patient-related factors which have not been included in the present study.


The results also indicate that in there is no statistically significant difference between both groups . Two variables had significant differences between elite and professional players  and moving the leg opposite the throwing arm in space (p = 0.00)). To conclude, the article examines specific movement patterns of single side fake movement in elite players and the confirmed importance of efficient skill execution in top level handball. On the contrary, less skilled players use more space for the same technical element.Feint movement is an important factor for offensive players to outplay their guard, and score. So far, there is no evidence of feint biomechanical analysis on a sample of elite players in handball or other team sports. Therefore, this study aimed to investigate kinematic parameters of single side fake movement between elite and professional level handball players. The sample of participants consisted of 10 handball players divided into two subsamples: elite handball players  and professional handball players . The kinematic analysis was conducted using a GAIT—LaBACS software system. Variables consisted of two phases  of feint single change of direction. Both phases included seven kinematic parameters that were observed. Statistical analysis included descriptive statistic parameters. The differences between elite and professional handball players were analyzed by multivariate and univariate variance analysis. Results showed significant differences between elite and professional players (λ = 0.44,  Handball is a technical-tactically, physically and psychologically complex sport that requires full engagement from players ,2. FactoThere are many fake movements that attackers use to trick defenders, but most complex are feints with the ball. These movements are commonly used by handball players, but are lacking any scientific approach. The feint single change of direction belongs to the attacking technique with the ball. Rogulj et al.  define fA review of the aforementioned aspects leads to the assumption that the execution technique of those skills plays an important role in success determination. Moreover, apart from anthropology, motor and functional skills, individual technique plays an important role in differentiation of elite and non-elite players ,11,12. LSo far, the feint technique has been analyzed only as a sport-specific test  or in teHowever, there is no evidence of feint biomechanical analysis on elite players sample in handball or other team sports. Following all aforementioned, this study aimed to investigate kinematic parameters of single side fake movement between elite and professional level handball players. More specifically, step lengths, angles between joints and movement speed will be measured by kinematic analysis. The sample of participants consisted of 10 handball players divided into two subsamples: elite (representative) and quality (first league) players. The selection criteria for elite players was participation in at least five major top-level handball competitions . The selection criteria for quality players was participation in at least three seasons in the First Division National Championship. For the sake of time efficiency, all players were recruited from wider surroundings of the city of Split during the offseason period. Elite handball players (representative sample) were represented by five players who played for the Croatian national handball team and won medals in the Olympic Games, World and European handball championships . Professional handball players (first league sample) were represented by five players who have a minimum of three seasons in their handball career in the First Croatian Handball League . Informed consent was obtained from all subjects involved in the study. Experimental procedures were completed following the declaration of Helsinki. All athletes participated in handball training on a daily basis, which had a significantly higher risk than the testing procedure conducted. All of them were aware of minimal risk identified and voluntarily participated in the study. Hence, there was no need for ethical board approval.The kinematic analysis was conducted using a GAIT—LaBACS software system  designed at the Faculty of Electrical and Mechanical Engineering and Naval Architecture in Split . VariablFor the purposes of kinematic analysis, twelve symmetric reference points were defined: The reference point is placed at the lower end of the outer ankle ; the reference point is placed in the middle of the knee joint from the front at the chip position (patella); the reference point is placed at the top of the pelvic bone at the point of the (cresta iliaca); the reference point is set on the (acromion); the reference point is placed at the end of the ulnar bone (olecranon); the reference point is placed at the ends of the ulnar and radial bones from the anterior side . The recStatistical analysis included calculation of descriptive statistic parameters while normality was tested using the Kolmogorov–Smirnov test procedure. The differences in kinematic parameters between elite and professional handball players were analyzed by multivariate  and univariate variance analysis (used to determine differences between each parameter of the given fake movement phase). The level of significance was set at p < 0.05. The software Statistica ver.13.0  was used for all analysis.p > 0.20). The calculation of descriptive statistic parameters included means, standard deviations and minimum and maximum for both phases and groups.The Kolmogorov–Smirnov test exhibited normal distribution of the variables (p = 0.00), in fake phase . The results also indicate that in the second statistically significant difference between groups, do not exist .p = 0.02) and MOT (p = 0.00)). Elite players execute smaller steps (SLS) during the first phase of the fake movement (106.44 cm) than professional players (133.55 cm). MOT results implicate that elite players exhibit a lesser angle (17.70°) than professional players (31.42°).Univariate analysis of variance (ANOVA) is presented in Differences between groups in the second phase of the fake movement are presented in This study aimed to investigate kinematic parameters of single side fake movement between elite and professional handball players. The obtained results indicate several important findings: (1) Players differ in the first phase of the feint; (2) elite players have a smaller first step and move more sideways in the first phase; and (3) differences in the second phase are connected to first phase performance but are not statistically significant.The literature review refers to significant differences in movement pattern efficiency dependable on players’ skill levels. This was noticed between skilled and novice players in volleyball, soccer, basketball and tennis ,23,24,25Our results correspond to previously mentioned studies in terms of differences between elite and professional players, as some of kinematic parameters differ between the groups. Still, it was evident only in the first phase of the fake movement. Results show that elite players performed specific movement pattern with the shorter sidestep for 27.11 cm than professional players.Additionally, elite players conduct fake movements more to the side, unlike professional players who perform them more diagonally. This can be noticed from the MOT variable in which professional players during the fake movement have a larger angle of 13.72° between the feet and frontal plane. The diagonal position of the attackers’ feet puts the offensive player in less favorable position towards the defender. This way the attacker is closer to defender and has less space for executive phase of fake movement that should be performed faster and more explosively. In addition, by thrusting too far forward, the attacker leaves the defender more sideways, and in order to pass him on the other side in the next moment (which is the next phase of the feint), it is necessary to go “back” and then go around the defender. Again, we see that time is important for performing a successful feint. The defender’s job is to stop the attacker with tackles. In those tackles, he always tries to be as close as possible to the attacker . Hence, This study had several considerable limitations. Although players in two groups played the same playing position, we noticed a significant difference in anthropometrical indices that could influence skill execution. In several studies, positive correlations were noticed between the body mass and body height and throwing kinematic parameters. Body mass influenced speed variables while body height influenced the height of the body’s center of gravity ,28. SimiAdditionally, lack of some more precise anthropometric measurements could be influential in the assessment of feint movement. Furthermore, in this study we have researched only kinematic parameters of single side fake movement. Future studies should include other factors that are important in handball players’ feinting performance. Two aspects should be specially researched: (1) cognitive abilities of players, such as perceptual and decision-making factors, and (2) physical abilities of players, such as timing, muscle quality, strength, power and/or reactive agility factors. It would be important to know which of these to is more important for successful handball feint performance.Research of kinematic parameters of single side fake movement for feint single change of direction has not been carried out so far, not just in handball but in team sport games generally. Hence, it was impossible to compare the obtained results and methodology with literature review. Results show specific movement pattern of single side fake movement in elite players and confirmed importance of efficient skill execution in top level handball. On the contrary, less skilled players use more space for the same technical element. Results of the study could direct handball coaches in better understanding single side fake movement performance. During teaching this important element they should focus on: (1) optimal step length; players’ needs to adapt first step length to a defender’s position and his anthropometry, (2) maintaining a straight body position while shifting center of gravity; during teaching the single side fake movement strait body position is crucial for controlling body balance and good perception of the game and (3) controlling the distance from defender; conducting single side fake movement to close to defender is inefficient, so the attacker should be learned to avoid physical contact and perform it on 5 to 10 cm distance of the defender’s arms reach.


TRA/TRB) T cell receptors (TCRs) in IAR T cells from the blood of healthy, at-risk, new onset, and established T1D donors. Using TCR sequences as barcodes, we measured infiltration of IAR T cells from blood into pancreas of organ donors with and without T1D. We detected extensive TCR sharing between IAR T cells from peripheral blood and pancreatic infiltrating T cells (PIT), with perfectly matched or single mismatched TRA junctions and J gene regions, comprising ~ 34% of unique IAR TCRs. PIT-matching IAR T cells had public TRA chains that showed increased use of germline-encoded residues in epitope engagement and a propensity for cross-reactivity. The link with T cells in the pancreas implicates autoreactive IAR T cells with shared TRA junctions and increased levels in blood with the prediabetic and new onset phases of T1D progression.Human islet antigen reactive CD4 + memory T cells (IAR T cells) from peripheral blood have been studied extensively for their role in the pathogenesis of autoimmune type 1 diabetes (T1D). However, IAR T cells are rare, and it remains poorly understood how they affect T1D progression in the pancreas. Using single cell RNA-sequencing coupled with a multiplexed activation induced marker (AIM) enrichment assay, we identified paired TCR alpha/beta ( Levels of IAR T cells may be increased in the pancreas especially during the active phases of islet autoimmunity, which may last months to years before and after clinical diagnosis 3. Since pancreatic biopsy is not tenable in humans, most efforts have focused on peripheral blood. IAR CD4 + and CD8 + T cells are found in blood of at-risk and T1D subjects, but also often in healthy control (HC) subjects 10. Distinctive phenotypic properties of IAR T cells in T1D subjects suggest their association with disease, yet because IAR T cells are rare in the blood  9, it remains difficult to ascribe a role for them in pathogenesis in the pancreas.Many studies have investigated the role of islet antigen reactive (IAR) CD4 + and CD8 + T cells in peripheral blood of subjects with Type 1 Diabetes (T1D). Because of their specificity for islet autoantigens, IAR T cells are thought to be involved in autoimmune destruction of the pancreas. IAR T cells have been investigated for their role in disease mechanisms and as therapeutic targets and biomarkers for beta cell destruction 11. Using paired TCR sequences identified by single cell RNA-sequencing (scRNA-seq), we previously demonstrated expanded clones of IAR T cells in blood of T1D patients 12. More recently, we identified a population of expanded IAR T cells with diverse specificities determined by TCRs with restricted TRA junctions and germline-constrained antigen recognition properties 13. In combination, these previous studies suggest the possibility that expanded IAR T cell TCR sequences in the blood of T1D patients represent the ontogeny of T cell autoimmune responses during disease. While this suggestion is attractive for both fundamental and translational investigations in T1D, evidence that IAR T cells with these characteristics are found in the pancreas is lacking. Studies have characterized islet infiltrating T cells in the pancreas from organ donors with T1D 16, but their linkage to IAR T cells in the blood is unclear.A defining feature of T cells is the presence of T cell receptors (TCRs) on their cell surfaces. T cells proliferate in response to TCR recognition of antigenic peptides, resulting in clonal expansion of a population of cells with identical TCR sequences at both the nucleic acid and protein sequence levels and the same antigen specificity TRA chains with PIT TCRs, and vice versa. We characterize PIT-matching TCRs and show that they have features of germline-like (innate) mode(s) of epitope engagement that are associated with TCRs displaying multi-specificity. Finally, we show expansion of PIT-matching TRA chains in blood prior to and near the time of T1D diagnosis, strengthening the link between IAR T cells in blood and pancreatic destruction.In this study, we have examined IAR T cells in the pancreas by comparing TCR junction sequences between circulating IAR T cells and pancreatic infiltrating T cells (PIT). We show that a significant fraction of IAR TCRs in peripheral blood share matching in vivo expansion of IAR T cells seen in peripheral blood 12 reflects autoimmune destruction of the pancreas during T1D. This hypothesis predicts the presence of IAR T cells in the pancreas during disease. To test this prediction, we utilized the extreme sequence diversity of TCRs to enable their use as “barcodes” for clonal populations of T cells recognizing specific antigens 17. We reasoned that because of the sequence diversity of TCRs and the rarity of IAR T cells in blood, it would be unlikely for individual IAR T cell TCR sequences to be present at high levels in PIT cells by chance. Significant overlap between IAR and PIT cell TCR sequences would therefore suggest a biological role for IAR T cells in the pancreas.Our central hypothesis is that Table S1 and We utilized TCRs from IAR T cells isolated from the peripheral blood of two subject cohorts  in length, 12 AA overlap) from the above islet proteins (Table S1). For both groups, CD154 + islet peptide activated cells were magnetically enriched, isolated as CD154 + CD69 + by single cell flow sorting and subjected to scRNA-seq to identify paired TRA and TRB chains in IAR T cells. In most experiments, we used TCRs from Cohort 1, comprising 4,331 TCR junctions  from 2,784 cells and 53 donors (Tables S1 and S2). Where indicated, we combined Cohorts 1 and 2 and subset to donors with HLA-DRB*04 alleles, yielding 2,967 junctions  from 1,873 cells and 43 subjects (Table S1).We isolated IAR T cells from peripheral blood using an overnight activation induced marker (AIM) assay based on upregulation of CD154 and CD69 DRB1*0401 HLA class II alleles, while ~ 10% had DRB1*0301 alleles. Using molecular cloning, lentiviral re-expression, and TCR functional assays, we identified multiple specific islet antigen epitopes that triggered 29/47 (~ 62%) of the TCRs tested from this cohort 13. These TCRs, therefore, represented IAR T cells from peripheral blood with a wide range of specificities.For our initial experiments, we focused on Cohort 1. Approximately 90% of these subjects had high-risk 18; a protocol at Vanderbilt University Medical Center/University of Pittsburgh; the Integrated Islet Distribution program (IIDP); and the Alberta Diabetes Institute Islet Core (ADI). PIT cell TCRs comprised a total of 9,798 unique TRA and TRB junctions from pancreatic tissues of autoantibody negative donors without diabetes, autoantibody positive donors representing prediabetic/preclinical stages of T1D, and T1D donors . TCR junctions represented 4,706 T cells and 5,092 CD8 + T cells. PIT cell TCR sequences contained multiple perfectly matched amino acid junction sequences from preproinsulin reactive TCRs found in islets and peripheral blood 19. IAR T cell and PIT cell TCR sequences were subjected to several filtering steps before use in subsequent analyses  analyses  and are TRA junctions compared to TRB junction matches  of total IAR T cell TRA junctions perfectly matched PIT junctions; conversely, ~ 0.45%  of PIT cell TCRs matched IAR T cell TCRs. The distribution of perfect matches between different subject groups  and cell types  did not differ significantly from the distribution of total TCR populations from each group . Thus, there were numerous perfectly matched TRA junction sequences shared between IAR T cells and PIT TCRs, even though the PIT TCRs were from different individuals than the IAR T cells.To test for TCR overlap, we first utilized string matches to test for perfectly matched recombined V-J or V-D-J junction sequence overlap between IAR and PIT cell TCRs. Unless otherwise noted, sequence comparisons were made at the amino acid level. We reasoned that perfectly matched junctions are most likely to reflect conserved function. This test showed multiple perfectly matched junction sequences, mainly  matches . Because20. To compare IAR PIT-matches with those in the unselected repertoires, we first down-sampled the unselected repertoires to match the size of unique IAR TRA junctions . We then used Fisher’s exact test to compare numbers of perfectly PIT-matched to non-matched TRA junctions from IAR T cells versus unselected HC and COVID-19 T cells. We found more PIT matches with IAR TRA junctions than with junctions from unselected repertoires: there were 47 perfect matches with IAR junctions (2.9%) versus 22 (1.4%) for HC junctions. For IAR T cell junctions, this comparison yielded a log2 odds ratio > 1 (or > 2-fold in linear units), far outside the 95% confidence intervals (2 odds ratio was ~ 0 (or ~ 1 in linear units), and clearly within the 95% confidence intervals, for PIT-matched to non-matched junctions from COVID-19 patients versus HC subjects  ntervals   A plot of the numbers of PIT TRA junction matches with IAR versus HC T cells at different PIT mismatch thresholds . This led us to conclude that the low frequency of PIT matches in V gene regions was not attributable to peptide presentation by different HLA molecules.Since the TCR We obtained results in TRA matches were related to T cell expansion and T1D progression. As a test, we quantified levels of PIT TRA matches in IAR TCR junctions found in > 1 cell (expanded) versus non-expanded TRA sequences in a cross-sectional analysis by disease group, initially using the Cohort 1 data set. To ensure that results were not biased by unequal numbers of junctions between disease groups, we randomly down sampled to equal numbers of junctions per group. This preliminary analysis revealed that the fraction of PIT TRA junction matches with expanded IAR T cells was significantly elevated, and the fraction of PIT TRA junction non-matches reduced, in newT1D subjects, relative to HC and T1D subjects.We speculated that PIT 23, we repeated the analysis using Combined cohorts  from 43 donors (Tables S1 and S2). Since we were able to sample relatively few 1AAb and 2AAb HLA-DRB1*04 subjects in Cohort 2, we performed this analysis without correction for different sample sizes. In cells with expanded TCRs, we saw a progressive increase in the fraction of PIT-matched junctions in IAR T cells collected from 1AAb, 2AAb and newT1D donors, then a decrease in T1D subjects to a similar level as in HC donors . We observed the same trends as with junction frequency, though the effects were weaker and less significant. This suggests that these studies were underpowered at the donor (sample) level. Despite this limitation, these results supported the conclusion that an elevation of expanded PIT-matched IAR T cell TRA chains in blood occurred prior to the onset of clinical disease.To control for p-value inflation by increased numbers of junctions from relatively few donors, we repeated this analysis at the donor level  than non-matched junctions, as was expected by the different donor pools used to isolate IAR and PIT cells. Previously, we identified a population of IAR T cells with diverse specificities determined by TCRs with restricted TCR alpha junctions and germline-constrained antigen recognition properties 13. To determine how this population of public TCRs was related to PIT-matched/non-matched TRA junctions, we tested for junction sequence overlap. We found significant overlap between public IAR T cell and PIT-matched TRA junctions  but not private IAR T cell and PIT-matched TRA junctions . Thus, there was strong overlap between cell populations with public and PIT-matching TCRs.We hypothesized that PIT-matched TRA junctions was significantly left shifted (shorter) in PIT-matched junctions than in non-matched junctions  (TRA junctions had a median length of ~ 39 nucleotides (nt), versus a median length of ~ 42 nt for PIT-non-matched junctions  . Thus, PIT-matched TRA junctions, but not their paired TRB junctions, were shorter, and more hydrophobic than PIT-non-matched TRA chains.As another test of differences between PIT-matched and non-matched junctions, we compared sequence features that have been correlated with TCR autoreactivity 13,24,25.. We utilov test) . PIT-matference) . We did  ~ 42 nt . Furtherunctions , but notunctions , were moTRA junctions were keys to the function(s) of IAR T cells having these TCRs. To further elucidate these sequence features, we used IMGT/HighV-QUEST 27 to characterize individual TCRs from their nucleotide (nt) sequences. We first determined the positions of single AA mismatches between PIT and IAR T cell TRA junctions. Since both V and J gene regions are germline-encoded, we predicted that PIT mismatches would not localize to these regions. Instead, we predicted that mismatches would instead occur primarily in the V-J recombination region, which is non-germline-encoded and varies in sequence between different TCRs. We found that mismatched residues were distributed symmetrically around amino acid position 4 . By comparison, the C terminal AA from V genes (3’ end) and the N terminal AA of J genes (5’ end), which mark the boundaries of germline-encoded residues in the respective gene segments, were centered at AA residues 3 and 5, respectively . Quantitatively, ~ 88%  of PIT-matched TRA junction mismatches were located between the 3’ends of V genes and the 5’ends of J genes. Normalizing the site of mismatches for junction length reduced variability but did not change the essential features of the results. Thus, PIT mismatches occurred primarily in the non-germline-encoded V-J recombination region.We speculated that sequence features of PIT-matching 28. A comparison of CDR1 region lengths identified using IMGT/HighV-QUEST 27 showed that the median CDR1 length was identical in PIT-matched and non-matched junctions, but the distribution was left-shifted significantly  for PIT-matched junctions . CDR2 regions showed no length differences between PIT-matched and non-matched TRA chains , whereas CDR3 regions, as expected since they comprise most of the junction sequences compared in TRA chains , by ~ 3 nt (1 AA). In other analyses, we found that framework (FR) regions FR1, FR2, FR3 and FR4 did not differ in length between PIT-matched and non-matched TRA chains, showing selectivity of the differences in CDR1 and CDR3 lengths.We next focused our comparisons on regions important for TCR binding and function. Analogous to immunoglobulin molecules, TCR complementary determining regions (CDRs) convey the specificity for antigen and major histocompatibility complex molecules TRA chains. Since sequence variability of CDR3 regions mostly comes from random addition of nucleotides not encoded in the genome (N region), we compared N region lengths between the two groups of TRA chains. This comparison showed that PIT-matched junctions had median N region lengths of ~ 3 nt (1 AA) whereas PIT-non-matched junctions had median N region lengths of ~ 5 nt (1–2 AA) . We also quantified contributions of the V gene and J gene segments adjacent to the N region. The 3’ end of the V region was slightly shorter in PIT-matched than non-matched TRA junctions (10 versus 11 nt)  whereas the 5’ end of the J region was slightly longer (27 versus 25 nt) . Thus, the difference in CDR3 lengths was a complex product of genome- and non-genome-encoded regions in PIT-matched junctions. Together, these analyses showed that PIT-matched and non-matched TRA junctions differed in V gene regions CDR1 and CDR3 that are important for peptide binding.We wished to determine the source(s) of junction length differences between PIT-matched and non-matched TRA chains was enrichment for TRA V gene segments with shorter CDR1 regions. To test this, we compared enrichment of V gene segments in PIT-matched versus non-matched TRA chains . This showed that TRAV12–2*01 and TRAV41*01 V genes were significantly over-represented in PIT-matched IAR T cell TRA chains, whereas TRAV4*01 and TRAV26–2*01 were under-represented (or over-represented in PIT non-matched TRA chains). Over-represented V genes, TRAV12–2*01 and TRAV41*01, had CDR1 region lengths of 15 and 18 nt (5–6 AA). In contrast, under-represented V genes, TRAV4*01 and TRAV26–2*01, had 21 nt (7 AA) CDR1 regions. Thus, selective utilization of V genes with shorter CDR1 lengths was a likely explanation for enrichment for TRA V gene segments having shorter CDR1 regions in PIT-matched junctions.The difference in CDR1 region lengths was an unexpected result, since CDR1 regions are germline-encoded and not considered variable. We hypothesized that the basis for the shift in CDR1 length of PIT-matched 22. Based on this fact, we hypothesized that the observed length variation of these regions suggested altered peptide binding properties for PIT-matched TRA chains. As a test, we utilized molecular modeling to predict peptide binding residues in tri-molecular models of PIT-matched and PIT-non-matched TCRs complexed with peptide-class II MHC complexes. For modeling, we used TCRmodel2, which is based on the AI system, AlphaFold v2.3 29, a newly described method that predicts protein structure with high accuracy at unmatched scale 30. TCRmodel2 uses focused databases of TCR and MHC sequences to expedite multiple sequence alignment feature building; optimization of the TCR template selection; and utilization of peptide–MHC complex structures as templates to improve modeling accuracy. For model input, we used paired TRA and TRB sequences from a set of 30 IAR TCRs with known specificity (16 PIT-matched and 14 PIT-non-matched TCRs), together with their cognate peptides 13; and sequences of HLA DRA*01:01 and DRB1*0401 MHC class II subunits 31.CDR1 and CDR3 regions are involved in binding to peptides presented by MHC molecules TRA and TRB sequence features. We first visually compared TCR-peptide contact residues in representations of PIT-matched and PIT-non-matched TCRs with extremes in TRA junction length (Table S3). We displayed the numbers of contacts in different sequence features in both chains from each TCR as a function of TRA and TRB junction lengths . As expected, the TRA and TRB CDR3 regions contributed the most peptide contacts. There was a significant positive relationship between the number of peptide contacts mapping to the TRA CDR3 region and TRA junction length, and a weaker negative relationship with the TRB CDR3 region . While the number of contacts was low, there was also a negative relationship between the TRA CDR1 region and TRA junction length.From best fit models for each TCR, we identified likely peptide contact residues (TCR residues < 5 Å in distance from the bound peptide chain) and mapped these to n length –B. This TRA or TRB chains between the two groups , while maintaining the separation in length between PIT-matched and PIT-non-matched junctions J recombination.The variation in contact residues by junction length (and PIT match) could indicate either differences in overall number of contacts, or different ratios of contacts in different regions. To test these possibilities, we first compared the overall number of TCR contacts by TCR chain in PIT-matched versus PIT-non-matched TCRs. This comparison showed no significant difference in overall peptide contacts in either o groups . In contunctions . Thus, pTRA chains also showed a decrease in CDR1 region length . This incongruity could suggest a change in physical properties of the shorter CDR1 regions to make them more amenable to close peptide contacts. Thus, it may be important that PIT-matched CDR1 regions were weakly more hydrophobic than PIT-non-matched regions .Concomitant with the increase in the ratio of CDR1 to CDR3 contacts, PIT-matched TRA junctions would lead to altered peptide recognition properties, such as strength and/or specificity of binding. As a measure of binding strength, we compared the functional avidities of TCRs having PIT-matched and non-matched TRA junctions specific for different peptide epitopes as described previously 13. We plotted cell proliferation, as measured by CFSE 13, versus peptide concentration , the frequency of these multi-specific clones was higher in public compared with private clones 13. In the present studies, we noted that TRA chains from all three of these multi-specific TCRs were PIT-matching. Indeed, multi-specific TRA junctions were modestly more frequent in PIT-matching than non-matching TRA junctions .Based on these results, we next hypothesized that TCRs with PIT-matched and non-matched 13. In parallel and independent experiments, we unexpectedly found that the TRA chain of a TCR (P196–1) from Influenza A/MP54- reactive CD4 + T cells perfectly matched the TRA chain from Clones 81 and 566  recognized non-overlapping GAD65 epitopes  and 566 . Furtherposition . This seposition . We foun32 of the Clone 81 TCR 13 and complexes of these peptides together with HLA-DRA1*0101/DRB1*0401 molecules 31. The cognate GAD65 377–396  and MP74 97–116 (VKLYRKLKREITFHGAKEIS) peptides did not show compelling sequence similarity by multiple sequence alignment. They also showed no evidence of structure 33 or aromatic side chain 34 conservation characterizing “hotspots” of molecular mimicry used by some crossreactive TCRs. Despite their low degree of peptide sequence similarity, molecular models of Clone 81 TCR with both peptides yielded identical scores of 0.88. In addition, TRA and TRB chains in both models showed similar predicted topography in their interactions with peptide-MHC class II complexes . Overlapping but non-identical residues in the modeled TCR TRA and TRB chain CDR1, CDR2 and CDR3 regions contacted the different peptides . Alignment of predicted structures of the TRA  and TRB  chains from models made with the different peptides showed nearly perfect superposition except in the CDR3 regions. This suggests that the PIT-matched Clone 81 TCR accommodates quite different peptide sequences through interactions involving conformationally conserved CDR1 (and CDR2 chains for TRB) regions, together with more variable CDR3 regions.To elucidate possible mechanisms of cross-reactivity of the Clone 81 TCR for the MP54 97–116 and GAD65 377–396 peptides, we constructed molecular models TRA junctions differed in peptide binding specificity. Recognizing that the number of known multi-specific TCRs from IAR T cells was too small at present to enable firm population-based conclusions about multi-specificity, we took an alternative approach to test our hypothesis. In other experiments, we observed that VDJdb, a curated public database of TCRs with known specificities 35, contains TCR sequences that recognize both single and multiple peptide epitopes. While we recognized that these designations were likely biased, with some TCRs being designated as single specificity because they were understudied, and others being designated as multi-specific on the basis of reactivity with closely related peptides. We reasoned, however that these errors would tend to offset each other, and that the size and scope of this database would provide a more comprehensive source of specific and multi-specific TCRs, which could be used to test their junction sequence features. There were N = 17,826 unique TRA chains in VDJdb that recognized single epitopes and N = 1,669 TRA chains that recognized multiple epitopes  nucleotides (~ 1 nt in median length) than TCRs with single specificity . Thus, multi-specific TCRs from VDJdb shared multiple sequence features with PIT-matched TCRs, including shorter TRA but not TRB chains, that had fewer N region nt, and encoded more hydrophobic AA sequences.We wished to test in grerater depth our hypothesis that PIT-matched and non-matched epitopes . Since n regions  from TCRcificity . Finallyepitopes . None ofTRA chains with PIT TCRs, and vice versa. Thus, a subset of IAR T cells shares a TCR chain with TCRs present in the pancreas. Since there are no familial or genetic relationships between the Cohort 1 and Cohort 2 blood donors and the nPOD organ donors, the TCR chain sharing we demonstrate involves public sequences. We also show that frequencies of PIT-matching TRA chains in blood increase prior to the time of diagnosis, suggesting a temporal linkage of levels of PIT-matching TRA chains in blood with disease progression. Shared TRB chains between peripheral blood and insulin-reactive TCRs from the pancreas were reported recently 19. These combined studies place autoreactive TCR chains at the scene of disease (the pancreas) at the right time (early in the disease process) to influence disease development.It has long been unclear whether and how rare autoreactive cells in peripheral blood represent autoimmunity in in target organs. In other words, are these cells drivers or passengers in autoimmune processes? To help resolve this question, we show here that a significant fraction of IAR TCRs from peripheral blood share matching TRA chains shared quite divergent TRB chains, and it remains unknown what fraction of these TCRs recognizes autoantigens. Nonetheless, our demonstration of TRA chain sharing strengthens the link between IAR T cells in blood and autoimmune destruction of the pancreas.However, we must emphasize that, at present, we do not know how many of these autoreactive TCR chains are associated with functional autoreactivity versus β-cells. At present, there is no way to predict with certainty that TCRs share specificity unless they show complete identity of both chains. In the present case, PIT-matching 37. Paradoxically, TCR cross-reactivity is necessary because the diversity of TCRs, while huge, is dwarfed by the vast array of potential foreign peptide-MHC complexes 39. Cross-reactivity expands the potential range of a given repertoire towards foreign antigens, but it comes at the expense of greater potential for increased self-reactivity or autoimmunity. These considerations emphasize the importance of understanding of sequence and structural features determining TCR cross-reactivity. Unfortunately, at present such an understanding remains incomplete 40. Our data show that cross-reactive (multi-specific) TCRs share sequence features with PIT-matched TCRs, namely shorter and more hydrophobic TRA junctions and diverse TRB chains, reminiscent of a previous report in mouse 41. Supporting this conclusion, the few islet-specific TCRs for which we have demonstrated cross-reactivity are PIT-matched TCRs. Earlier work demonstrated shortened 25 and more hydrophobic 24TRB chains in bulk T cell subsets from T1D subjects, and suggested that these features are important in development of self-reactive T cells.T cell specificity is key to cellular immunity 22. For most TCRs, TRB CDR3 contacts are most numerous 42, although there are multiple examples of MHC class I-restricted TCRs that predominantly utilize germline-like TRA CDR3 regions 47. Less is known about such TRA-centric, MHC class II-restricted TCRs, such as those we have observed. One of the V genes enriched in PIT-matched TRA chains , TRAV12–2, is also a feature of multiple MHC class I-restricted TCRs 47. The other V gene enriched in PIT-matched TRA chains was TRAV21–2, which is associated with celiac disease, while paired with TRBV9 chains Our findings suggest that utilization of TRA-centric binding is a previously undescribed but widespread feature of IAR TCRs, and perhaps MHC class II-restricted TCRs in general.TCR CDR3 regions form most of the primary contacts with peptide epitopes presented by MHC molecules 46. This feature may contribute to the high precursor frequency and immunodominance of this TCR 46. PIT-matching IAR T cell TRA chains also employed less non-templated and more germline-like (innate) mode(s) of epitope engagement. TRA CDR3 regions of PIT-matching IAR varied from the genome by relatively few N region nucleotide sequences. In addition, PIT-matching IAR T cell TRA chains showed shorter CDR3 regions and longer TRAJ segments than non-matching IAR T cell TRA chains. TCR CDR1 regions contact both peptide epitope and MHC residues, while CDR2 regions generally contact only MHC residues 22. Thus, TCR regions important for peptide binding were shortened in PIT-matching TRA chains and had altered peptide binding properties. These PIT-matching TRA chains were also more hydrophobic at the amino acid level, suggesting partner agnostic interactioins via the hydrophobic effect versus more residue specific effects such as hydrogen bonding. Our results also suggest that these shorter and more hydrophobic, germline-like, TRA chains are a feature of many cross-reactive TCRs. While some TCRs that predominantly utilizing germline-like TRA CDR3 regions are cross-reactive 49, this has not been previously demonstrated on the global level we show here.Molecular modeling showed that germline-encoded CDR1, rather than CDR3, residues from TRAV12–2 contributed critically to binding of an immunodominant TCR to Yellow fever virus 18, including spleen and pancreas draining lymph nodes, from the same donors used for PIT TCR identification. Another weakness in our studies is that it is underpowered with respect to numbers of subjects studied. This is apparent in our examination of individual donor frequencies between PIT-matched and non-matched TRA junctions . Our study is also underpowered for autoreactive TCRs of known specificity needed to directly test our hypothesis that PIT-matched TCRs tend to be multi-specific. Such a test is not possible at present because the numbers of IAR T cell TCRs with demonstrated specificity remains small. While we used VDJdb sequences of different specificities as an indirect test, a. more comprehensive collection of autoreactive TCRs with known specificity will be needed to further clarify the situation.One caveat to our study is that we have addressed only sharing of public TCRs. This was by necessity, since TCR sequences from IAR and PIT T cells from the same individuals were not available. While this feature enhances broadness of potential translational applications, it also limits broad mechanistic conclusions. Future studies may be able to address this weakness by testing of additional nPOD tissues TRA sequence features in both the pancreas and blood in early stages of T1D progression. Together, these new findings suggest that an alternative to the reductionist approach for translational studies is to consider autoreactivity as a web of T cell specificities and potential cross reactivities. Our findings provide a basis for expansion of future biomarker studies to include a broader network of autoreactive and potentially multi-specific TCRs.Our studies have potential translational implications. Typically, studies on TCRs as biomarkers have taken a reductionist approach by focusing on one or a few TCRs to monitor disease progression and/or response to therapy 19,50,51. In contrast to this approach, our broader-based studies place many TCRs with different islet antigen specificities and distinct https://github.com/BenaroyaResearch/. Profiles yielding TCRs were deposited in the GEO repository (https://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE182870 (Cohort 1) and GSEXXXXX (Cohort 2).Code and data for generating Figures, as well as pdb files of molecular models, are available at NCT00129259 for the AbATE trial 52 and NCT00515099 for the START study.53Protocols for these studies were approved by the Institutional Review Board of Benaroya Research Institute (IRB7109–332). Protocols for the clinical studies were approved under the auspices of Methods are available as Supplemental Methods.