Type 1 diabetes (T1D) results from immune-mediated destruction of insulin-producing beta cells. Prevention efforts have focused on immune modulation and supporting beta cell health before or around diagnosis; however, heterogeneity in disease progression and therapy response has limited translation to clinical practice, highlighting the need for precision medicine approaches to T1D disease modification.To understand the state of knowledge in this area, we performed a systematic review of randomized-controlled trials with We identify and summarize 75 manuscripts, 15 describing 11 prevention trials for individuals with increased risk for T1D, and 60 describing treatments aimed at preventing beta cell loss at disease onset. Seventeen interventions, mostly immunotherapies, show benefit compared to placebo (only two prior to T1D onset). Fifty-seven studies employ precision analyses to assess features linked to treatment response. Age, beta cell function measures, and immune phenotypes are most frequently tested. However, analyses are typically not prespecified, with inconsistent methods of reporting, and tend to report positive findings.While the quality of prevention and intervention trials is overall high, the low quality of precision analyses makes it difficult to draw meaningful conclusions that inform clinical practice. To facilitate precision medicine approaches to T1D prevention, considerations for future precision studies include the incorporation of uniform outcome measures, reproducible biomarkers, and prespecified, fully powered precision analyses into future trial design. Type 1 diabetes (T1D) is a condition that results from the destruction of a type of cell in the pancreas that produces the hormone insulin, leading to lifelong dependence on insulin injections. T1D prevention remains a challenging goal, largely due to the immense variability in disease processes and progression. Therapies tested to date in medical research settings  work only in a subset of individuals, highlighting the need for more tailored prevention approaches. We reviewed clinical trials of therapies targeting the disease process in T1D. While the overall quality of trials was high, studies testing individual features affecting responses to treatments were low. This review reveals an important need to carefully plan high-quality analyses of features that affect treatment response in T1D, to ensure that tailored approaches may one day be applied to clinical practice. Felton et al. conducted a systematic review to evaluate studies testing disease-modifying therapies and features linked to treatment response for type 1 diabetes prevention. While the quality of prevention and intervention trials is found to be high, precision analyses on factors associated with treatment response are of poorer quality. Since the discovery of insulin over a century ago, treatment options for persons with type 1 diabetes (T1D) have shown remarkable advancements, including improved insulin formulations, delivery methods, and tools to monitor glycemia2. Even with these transformative advances, considerable negative impacts remain on health outcomes and quality of life5. In contrast, effective disease-modifying therapies aimed at the preservation of endogenous insulin production could not only improve these outcomes but also, if given early enough in the disease course, prevent the need for insulin replacement9. Because T1D is an autoimmune disease, many agents tested as potential disease-modifying therapies are immunomodulatory, while others target pathologic contributors such as glucose toxicity and beta cell health and function10. In 2022, the US Food and Drug Administration approved teplizumab, a monoclonal antibody targeting CD3, as the first therapy to delay the onset of clinical T1D in at-risk individuals11.Type 1 diabetes (T1D) results from immune-mediated destruction of pancreatic beta cells12. The first ADA/ EASD Precision Medicine in Diabetes Consensus Report defined precision prevention as “using information about a person’s unique biology, environment, and/or context to determine their likely responses to health interventions” and states that “precision prevention should optimize the prescription of health-enhancing interventions”13. Given that agents targeting these pathways may have potential adverse effects, and initial therapies may affect the efficacy and safety of subsequent treatment approaches, precision medicine is uniquely poised to identify which individuals stand to benefit the most from a given intervention and to optimize potential risk-benefit ratios for treated persons. Additionally, once further T1D disease-modifying therapies are approved for clinical use, precision medicine will facilitate the selection of therapies guided by the individual’s disease, including potential combination regimens of disease-modifying therapies15.The Precision Medicine in Diabetes Initiative (PMDI) was established in 2018 by the American Diabetes Association (ADA) in partnership with the European Association for the Study of Diabetes (EASD). The ADA/EASD PMDI includes global thought leaders in precision diabetes medicine who are working to address the burgeoning need for better diabetes prevention and care through precision medicine. This Systematic Review is written on behalf of the ADA/EASD PMDI as part of a comprehensive evidence evaluation of precision prevention in T1D in support of the 2nd International Consensus Report on Precision Diabetes Medicine8. Based on this, in 2015, the diabetes research community adopted a staging system, with the development of multiple islet autoantibodies now heralding Stage 1 T1D9. At the time of clinical T1D diagnosis, insulin replacement is required but endogenous insulin production, though diminished, can still be detected in most affected individuals. While an ideal goal is clinical T1D prevention, disease-modifying agents aimed throughout the spectrum of T1D progression have the potential to improve long-term outcomes7. Furthermore, given widely available participants and shorter total trial durations, agents planning to target earlier stages of the disease are often initially trialed in the new-onset period16. Therefore, we sought to understand the current state of knowledge regarding precision approaches to T1D disease modification, either to prevent the development of early-stage or clinical T1D (referred to as “prevention” studies) or to preserve endogenous insulin function around the time of clinical T1D diagnosis (referred to as “new-onset” studies). Specifically, we asked if individual characteristics have been robustly identified to select persons for therapeutic optimization of T1D disease-modifying therapies before or at the time of diagnosis. We reviewed and summarized existing trials in this area and identified individual characteristics associated with treatment effects.T1D development occurs along a spectrum of progressive beta cell destruction, beginning with loss of tolerance, reflected by the appearance of islet autoantibodies, and continuing with progressive hyperglycemia, abnormal glucose tolerance, and decline in endogenous insulin production, reflected by a decline in C-peptideWe developed a search strategy using an iterative process that involved Medical Subject Headings (MeSH) and text words. This search was refined based on a sensitivity check for key articles identified by members of the group . Eligible study types included randomized controlled trials (RCTs); systematic reviews or meta-analyses of RCTs, or post hoc analyses of RCTs. Selected primary trials or longitudinal follow-up papers of primary trials had a total sample size ≥50 and were published as a full paper in English in a peer-reviewed journal within 25 years of the search (2/21/1997-2/22/2022). Papers focusing on a precision approach to identify features associated with a treatment response were also included if the total sample size was ≥10. Longitudinal follow-up papers of RCTs were included if they addressed follow-up data on time to diabetes, C-peptide area under the curve (AUC), or included “precision analyses” of specific individual features or measures of treatment response. Studies were excluded if they included mixed participant populations  or populations with inconsistent definitions across papers . Several additional key articles previously known to the group of experts that also met inclusion criteria but were not included in the search results because of search restrictions designed to improve search feasibility were also included in the analysis. While systematic reviews and meta-analyses were included in the original search strategy to identify any existing meta-analyses aimed at precision approaches, none that met inclusion criteria were identified. All included papers were primary trials or post hoc analyses of primary trials.Investigators independently screened and reviewed each potentially relevant article according to preliminary eligibility criteria determined by members of the review team. For Level 1 screening two investigators per article screened each title and abstract. Discordant assessments were discussed and resolved by consensus or arbitration after consultation with a member of the review leadership team . For Level 2 screening of eligible articles, full texts were retrieved and reviewed by two independent reviewers using the inclusion/exclusion criteria. Discordant assessments were similarly discussed and resolved.Two separate investigators per article extracted data from each article meeting inclusion criteria, with consensus determined by a member of the leadership team. Extracted data included study and publication name and date, if the study was single or multicenter, participant characteristics , eligibility criteria, intervention details, details of metabolic monitoring, duration of follow-up, primary trial outcome, specific data on outcomes of intervention on time to diabetes  or C-peptide (at furthest reported timepoint from treatment), and methods and findings surrounding precision analyses focused on treatment response.The protocol for this review was registered (PROSPERO ID: CRD42022310063) before implementation and amended during review to edit group members and for feasibility, to add further exclusion criteria .Investigators also independently performed quality assessments using Covidence’s Cochrane Risk of Bias template in tandem for each eligible study; this included assessments of sequence generation, allocation concealment, masking of participants/personnel, masking of outcome assessment, incomplete outcome data, selective reporting, and any other sources of bias to order to determine the overall risk of bias.Because of the heterogeneity of clinical interventions , we were unable to perform a meta-analysis but instead completed summaries of relevant studies. A forest plot was generated using hazard ratios from all included prevention studies. No studies were missing data or required data conversion for this. Reproducibility was ensured by a dual investigator review of each article at each review stage.Further information on research design is available in the 31 and in individuals with new-onset T1D 91.From 1006 studies identified by PubMed and Embase searches, 75 were eligible for extraction  antigen therapy was limited to individuals who were GAD autoantibody positive19. Most prevention trials  were multicenter studies; 9/11 (82%) were also double-masked, while 2/11 (18%) had no masking. In addition to these 11 papers, two follow-up papers and two papers focused solely on precision analysis of treatment response were also identified . Overall, only two prevention studies reported a positive impact on time to islet autoantibody positivity or time to diabetes: the primary prevention study testing whey-based hydrolyzed vs. cow’s milk formula30 and the secondary prevention study testing teplizumab21 , co-primary outcome in 2/43; 5%), although other studies used HbA1c and/or insulin dose and one study used T1D remission. Primary outcome was not specified in 5 trials. All follow-up studies focused on a measure of beta cell function. Trials reporting a measure of beta cell function as the primary outcome most commonly utilized mean C-peptide AUC from a mixed meal tolerance test; values for these data were available for 32/35 primary trials and 5/6 follow-up studies and are summarized in Supplementary Table To determine whether there were specific individual features that impacted response to treatment , we assessed papers that included this type of precision analysis. Two papers from prevention and 12 papers from new onset studies focused solely on precision analyses of treatment response . An additional 43 papers also included some aspect of precision analysis  of papers studied Fig. . PrespecBecause sample sizes inevitably decrease as groups are subdivided for precision analyses, we next looked at sample sizes for the precision subgroups. Only slightly over half (30/57) of papers reported sample sizes for all subgroups defined by precision features. Within these 30 manuscripts, we observed wide variability in the sample sizes of the subgroups analyzed. Figure Figure 87. Here, younger age groups showed improved treatment responses to teplizumab, ChAglyCD3, and Vitamin E. In contrast, older age was linked to a beneficial treatment response vs. placebo with high-dose antithymocyte globulin (ATG) and oral insulin 48. One study showed that younger age was linked to a more rapid decline of C-peptide compared to placebo in Bacillus Calmette-Guerin (BCG) vaccine-treated individuals32. Baseline measures of beta cell function were linked to differences in treatment response in 10/26 (38%) of analyses where this relationship was tested89. In two papers focused on prevention studies, measures linked to worsened beta cell function were associated with an improved response to treatment 24. Analyses testing trials in the new-onset period had split results: teplizumab, ChAglyCD3, linomide, and atorvastatin performed better compared to placebo in groups with better baseline beta cell function measures88. In contrast, canakinumab, imatinib mesylate, and the anti-IL-21/liraglutide combination showed stronger treatment effects in individuals with lower baseline beta cell function measures89. Taken in aggregate these results highlight evidence that baseline beta cell function may impact treatment response, but the direction of impact likely varies by treatment used and stage of disease.We next examined the types of features tested for relationships with treatment response Fig. . In triaInterestingly, in contrast to primary trial papers, precision papers most commonly tested relationships of an immune cell phenotype with treatment response (57%). Because only two papers identified included a favorable response to time to type 1 diabetes diagnosis, treatment response was assessed using a range of alternative outcomes Fig. . For allA finding impacting studies in all categories was a lack of racial and ethnic diversity in participant populations. Data on participant race were available in less than a third (23/75) of total papers; for reported papers, participants self-reporting as white race comprised a median of 92% of the total study population (interquartile range of 88-96%). Ethnicity was reported in 20 papers; within these manuscripts, participants self-reporting as identifying with a Hispanic ethnicity comprised a median of 5% of study participants (interquartile range of 3–9%).When assessing additional risks of bias, we found that many papers did not include details sufficient to assess these risks Fig. . AlthougWe filled a gap in the T1D literature by systematically reviewing 25 years of large randomized controlled trials focused on T1D disease-modifying therapies, as well as precision analyses focused on identifying features of treatment response. Several themes in the literature were identified. Immunotherapies were the most common disease-modifying agents tested, and a resounding majority of these agents were tested in “new-onset” trials after a clinical diagnosis of T1D. Of the 17 interventions that showed benefit in slowing T1D progression or preserving endogenous insulin secretion, only two were tested prior to clinical disease onset. Primary trial outcome papers most commonly included precision analyses testing the impacts of baseline age and beta cell function on treatment response, while post hoc precision analysis papers primarily focused on immune phenotypes.14. Along these lines, trials designed to limit participant heterogeneity based on features associated with treatment response could theoretically allow for clearer determinations of effect and a greater number of positive trials. While trials were overall of high quality, a key take-home message is that the current review did not identify high-quality clinical trial data supporting the existence of individual features consistently linked to therapeutic response and justifying translation to clinical care. Below, we highlight important considerations identified by this analysis for the future applicability of precision medicine to T1D disease modification.Based on clinical heterogeneity observed in phenotypes of T1D progression and severity, a precision-based concept that has gained popularity is the idea of the T1D “endotype”, a T1D subtype “defined by a distinct functional or pathobiological mechanism ”92. However, precision endpoints were much more variable and would benefit from a similar consensus definition of “responders” to disease-modifying agents within larger trial populations. Strategies applied have included time to diabetes, insulin use, stratification based on changes in C-peptide, and identification of individuals exhibiting less C-peptide loss compared to placebo67. Although C-peptide was by far the most frequent outcome measure used to identify differential treatment responses, approaches to stratify based on C-peptide were highly variable. Consistent approaches, such as a quantifiable metric based on expected values93 will allow better comparison of features associated with treatment response across trials.Time to T1D was the most consistent primary outcome of T1D prevention studies, while the vast majority of new-onset studies used mixed meal-stimulated C-peptide AUC, consistent with the consensus recommendations by Palmer and colleagues87. The association of age with treatment response is in keeping with the strong associations of age to features of T1D in many observational and natural history studies, before and after clinical diagnosis96. Differences in pancreas histology have been identified in donors with younger age of diagnosis98. However, it is unclear whether differences in treatment response linked to age are associated with differences in underlying disease pathophysiology vs. differences in severity or progression of T1D at the time of treatment. The observation that age differentially impacts outcomes in different trials, in addition to stratification of both immune phenotypes and beta cell function by age, supports the idea that the underlying biological reasons for age associations could be linked to mechanisms and are important to consider in future trial designs and potentially in future precision therapy.Age and measures of beta cell function were most frequently identified as factors associated with differential treatment response in primary trial and primary trial follow-up papers. For example, younger age was linked to improved treatment response in several new-onset trials using CD3-based agents106. Interestingly, findings somewhat differed depending on the stage of intervention. Here, two unique prevention studies testing oral insulin and teplizumab showed that worse beta cell function was associated with improved treatment outcomes compared to placebo24. In contrast, CD3-based therapy trials after disease onset showed an association between higher baseline insulin secretion and improved outcomes61. These differences highlight the importance of considering the disease stage in the design and interpretation of intervention efforts107. Especially at earlier stages in the disease process, abnormalities in beta cell function could allow insight into a therapeutic window during active disease or immune attack, and optimal timing of therapy108. In contrast, in more advanced diseases after diagnosis, associations with differences in beta cell function could reflect differences in the degree of disease progression, and so amenability to prolonged preservation of a larger residual beta cell mass. Differences in the relationships between beta cell function measures and outcomes for different agents in the new onset period also highlight agent mechanism of action as a critical consideration for designs incorporating beta cell function into the stratification of trial populations and precision approaches to disease-modifying therapy.Thirty-eight percent of studies testing the impacts of baseline beta cell function showed a significant link to treatment response, consistent with the substantial body of literature identifying an ongoing dialog between autoimmunity and the beta cell in T1D109. If novel mechanistic markers  can be used to predict treatment response, then similar scrutiny and standardization of these markers will be needed for cross-study comparisons and successful implementation.Specific autoantibodies and immune cell phenotypes were also linked to treatment response for multiple agents. An important consideration in these types of assays is reproducibility. The T1D field has been strengthened by an international standardization program for autoantibody measurement that underpinned the development of T1D staging criterian = 2 participants. Papers also tended to show positive results, raising concern for publication bias.Our review identified important methodologic considerations with many precision analyses. While there were multiple notable and interesting results, most trial manuscripts (primary or follow-up) included precision analyses that were not prespecified, which decreased the quality ranking of these studies. Corrections for multiple comparisons were rare. Additionally, subgroup sizes were infrequently reported, but when available, these group sizes were highly variable and as small as 24. Interestingly, within this trial, a significant protective effect of oral insulin was identified as part of a prespecified precision analysis of individuals with lower first-phase insulin response. Testing in future studies will be needed to understand the reproducibility of this finding. Another example of the application of this approach moving forward is the DIAGNODE 3 study (NCT05018585), which did not meet inclusion criteria for the current review but will prospectively test for a preferential benefit of GAD-alum injections in the HLA DR3-DQ2 population that was identified in post hoc analyses110. Based on frequent testing and existing studies suggesting impacts of baseline age and beta cell function as potential features associated with treatment response, pre-specified analyses for appropriately powered studies testing the impact of these precision features should be considered in trials moving forward.While these issues are a known limitation of hypothesis-generating exploratory analyses, follow-up studies focusing on testing positive findings a priori will be critical to the application of clinically meaningful precision medicine. An example of the necessity of hypothesis testing was the TrialNet oral insulin prevention study, which was prospectively designed to test a responder subgroup identified in the Diabetes Prevention Trial Type 1 (DPT-1) trial with high insulin autoantibody titers, and ultimately found no significant impact of treatment within this group111, led to a revision of the definition of T1D to include early stages of disease112. Stage 1 and stage 2 T1D are now defined by the presence of multiple autoantibodies without or with dysglycemia, whereas clinical disease is now considered as stage 3 T1D113. Because these stages were developed concurrently with many of the trials included in this review, these definitions were not applicable at the time of many of these trials, limiting our ability to apply staging categories to this review. As noted above, the timing of T1D stages impacts study feasibility  and may be critical to intervention efficacy, highlighting the importance of considering the disease stage in the design and interpretation of intervention efforts. Moving forward, widespread adoption of the T1D staging system combined with increased screening efforts spurred by the recent positive teplizumab trial in stage 2 T1D may allow for increased numbers of trials in earlier stages of the disease.Time to T1D was the most consistent primary outcome of T1D prevention studies, but inclusion criteria for these studies varied widely across trials, including combinations of genetic risk, presence of islet autoantibodies, and changes in glycemia and/or beta cell function. Recent progress in understanding the natural history of T1D, particularly the high lifetime risk associated with progression from multiple autoantibodies to clinical T1D115 and verapamil116. A large pediatric follow-up trial testing verapamil (positive outcome) and tight metabolic control with a hybrid closed loop (negative outcome) was published after the conclusion of our systematic review118. In addition, most studies reviewed did not report data on race or ethnicity. For those that did report these data, populations studied largely identified as non-Hispanic white. Barriers to screening of traditionally underrepresented populations is a recognized issue amongst T1D natural history and intervention studies119. This is especially important to address moving forward given the rising incidence of T1D in these populations120.This study has several limitations. The heterogeneity of included papers limited our ability to perform metanalysis. For feasibility, we restricted our review of primary trials to those enrolling a minimum of 50 total participants. Because of this, some trials were not reviewed, including positive trials testing alefaceptIn summary, our review identified noteworthy progress towards defining effective disease-modifying therapies for T1D but a need for better quality data to support the existence of individual features consistently linked to differences in treatment response. Our findings specifically highlight the need for standardization of precision outcome measures, reproducible biomarkers of disease pathology, and prespecified, adequately powered precision analyses. Reports of future trials would benefit from including adequate details to assess potential risks of bias.Supplementary InformationSupplementary Data 1Supplementary Data 2Description of Additional Supplementary FilesPeer Review FileReporting Summary


Single-nucleotide polymorphisms in genes controlling lipid mediator signaling may modify the colorectal polyp prevention activity of aspirin. Further investigation is required to determine whether testing for genetic variants can be used to target cancer chemoprevention by aspirin to those who will benefit most. 2. We investigated whether 35 SNPs in oxylipin metabolism genes such as cyclooxygenase (PTGS) and lipoxygenase (ALOX), as well as 7 SNPs already associated with colorectal cancer risk reduction by aspirin , modified the effects of aspirin and EPA on colorectal polyp recurrence in the randomized 2 × 2 factorial seAFOod trial. Treatment effects were reported as the incidence rate ratio (IRR) and 95% confidence interval (CI) by stratifying negative binomial and Poisson regression analyses of colorectal polyp risk on SNP genotype. Statistical significance was reported with adjustment for the false discovery rate as the P and q value. 542 (of 707) trial participants had both genotype and colonoscopy outcome data. Reduction in colorectal polyp risk in aspirin users compared with nonaspirin users was restricted to rs4837960 (PTGS1) common homozygotes , rs2745557 (PTGS2) compound heterozygote-rare homozygotes , rs7090328 (ALOX5) rare homozygotes , rs2073438 (ALOX12) common homozygotes , and rs104522 (TP53) rare homozygotes . No modification of colorectal polyp risk in EPA users was observed. In conclusion, genetic variants relevant to the proposed mechanism of action on oxylipins are associated with differential colorectal polyp risk reduction by aspirin in individuals who develop multiple colorectal polyps. SNP genotypes should be considered during development of personalized, predictive models of colorectal cancer chemoprevention by aspirin.Aspirin and eicosapentaenoic acid (EPA) reduce colorectal adenomatous polyp risk and affect synthesis of oxylipins including prostaglandin ESingle-nucleotide polymorphisms in genes controlling lipid mediator signaling may modify the colorectal polyp prevention activity of aspirin. Further investigation is required to determine whether testing for genetic variants can be used to target cancer chemoprevention by aspirin to those who will benefit most. Helicobacter pylori infection; ref. A large body of evidence has accumulated from epidemiologic observations, randomized colorectal polyp prevention trials, and long-term follow-up studies of colorectal cancer outcomes after participation in vascular prevention trials, that regular aspirin use is associated with a 15% to 20% reduction in colorectal cancer risk . However2 metabolite, PGE-M, as a predictive risk, or therapeutic response, biomarker of colorectal polyp prevention by aspirin  of personalized aspirin efficacy that could pinpoint individuals who are either sensitive to, or resistant to, the anti–colorectal cancer activity of aspirin . There h aspirin , althoug aspirin .Prostaglandin G/H Synthase (PTGS); also known as Cyclooxygenase (COX)-1 and -2] in post hoc analyses of randomized colorectal polyp prevention trials of aspirin has demonstrated a possible interaction between aspirin and PTGS2 SNP rs4648310  use . Investidatasets .2 from the omega-6 polyunsaturated fatty acid (PUFA) COX substrate C20:4n-6 arachidonic acid -mediated synthesis of other oxylipins with antitumorigenic activity such as leukotriene (LT) B4  B4 .HPGD), the enzyme responsible for inactivating PGE2, has been shown to predict colorectal cancer risk reduction by aspirin  and the effect of aspirin on colorectal polyp risk has not been addressed previously in a randomized trial setting.Despite the relevance of other genes controlling oxylipin synthesis for the chemopreventative property of aspirin, the relationship between SNP genotypes in a wide range of genes controlling oxylipin synthesis and levels (including n-3 eicosapentaenoic acid (EPA) also has colorectal cancer chemoprevention activity  at screening colonoscopy in the English Bowel Cancer Screening Programme  of the anticancer activity of aspirin and EPA, as well as SNPs in selected genes already linked to modification of colorectal cancer risk reduction by aspirin, and colorectal polyp outcomes in the seAFOod polyp prevention trial.This study is part of a wider program of investigations using the seAFOod trial biobank and post-trial BCSP colonoscopy outcomes called STOP-ADENOMA (ISRCTN05926847). The study was conducted in accordance with the Declaration of Helsinki. Ethical approval for this study was granted by London and Surrey Borders Research Ethics Committee (19/LO/1655). The seAFOod Trial biobank has been described elsewhere . Blood sDNA was extracted from a single buffy coat extract per seAFOod trial participant using an in-house phenol-chloroform method, followed by washing with absolute ethanol and storage at 4°C in DNase/RNase-free water, prior to spectrophotometric assessment of the DNA quantity and quality. If any sample had a DNA yield <20 ng/μL, a second sample for that individual was obtained from the Biobank for DNA extraction.Genotyping was carried out using the Fluidigm microfluidic SNP genotyping system (Fluidigm), using custom-built SNP Type genotyping assays (Fluidigm) for 92 SNPs (Supplementary Table S1).A multiplexed preamplification PCR was carried out to increase the available template DNA and reduce allelic drop-out associated with low template concentration. DNA samples were run on 96.96 Dynamic Array IFCs (integrated fluidic circuits), which were primed and loaded using the IFC Controller HX (96.96 arrays), as per manufacturer's instructions. In total, 691 individual samples including 10 technical duplicates, 31 participant duplicate samples and 1 participant triplicate sample, were analyzed, representing 648 individual participant DNA samples.Genotyping PCR and measurement of endpoint fluorescent values were carried out using the Fluidigm BioMark HD system. Data were analyzed and genotyping calls made using Fluidigm SNP Genotype Analysis software.Fourteen SNPs failed quality control; one SNP was monoallelic, one SNP was repeatedly called differently in duplicate sample runs, and 12 SNPs had an absence of genotype call >5% (Supplementary Table S1).P < 0.05 with Benjamini–Hochberg correction for multiple testing). However, on inspection of scatter plots produced during Fluidigm genotyping, “true calls” did not overlap with “fails” suggesting that this was not caused by sampling error. Therefore, these SNPs were included in subsequent analyses to avoid missing a relationship with clinical outcomes given that these SNPs could be causally related to colorectal polyp development  explaining absence of Hardy–Weinberg equilibrium.Therefore, genotype data for 78 SNPs were available for analysis inside the seAFOod trial database. Twelve SNPs did not satisfy Hardy–Weinberg equilibrium , as well as 7 SNPs previously associated with differential colorectal cancer risk reduction in aspirin users -funded Fatty Acid Metabolism (FAME) Consortium - Interlinking Diet with Cardio-metabolic Health. Briefly, all SNPs in PTGS and ALOX genes were grouped into linkage disequilibrium (LD) blocks using PLINK v1.9 software  with blood and rectal mucosal levels of PUFAs measured in the seAFOod polyp prevention trial, as well as colorectal polyp risk, are the subject of a separate report.R2 values for paired SNP relationships.Linkage disequilibrium between SNPs in individual genes was analyzed using the LDmatrix tool (NIH) to derive χ2 test or Mann–Whitney U test, as appropriate, to confirm similarity with the original seAFOod trial cohort and ensure continuing balance across the trial treatment allocations.Only seAFOod trial participants, for whom there were trial colonoscopy outcome data, were included in this SNP analysis. Baseline characteristics  of the seAFOod trial participants included in the SNP analysis were analyzed using the Interactions of SNP genotypes with trial interventions were analyzed “at the margins” , consistent with the primary seAFOod trial analysis  polyp number per participant  between colorectal polyp number and intervention for some strata, an interaction test between the SNP and intervention was conducted fitting the negative binomial regression model with additional terms for interaction between the treatment and SNP variables.All SNPs were then used to stratify a negative binomial regression model of colorectal polyp number  and two colorectal polyp outcomes, the positive false discovery rate (pFDR) was described by the q value for each of the models including individual SNPs .A total of 666 trial participants had at least one buffy coat sample vial in the seAFOod trial biobank . There wA total of 542 seAFOod trial participants, for whom SNP genotype data were available, also had colonoscopy outcome data from the seAFOod trial. Reasons for lack of primary  outcome data for some trial participants are detailed in the seAFOod trial CONSORT diagram . The cliPTGS1, PTGS2, and HPGD SNP revealed that PTGS1 rs4837960 common homozygotes (G:G) and PTGS2 rs2745557 compound heterozygotes (G:A)-rare homozygotes (A:A) were associated with a statistically significant reduction in total colorectal polyp number in aspirin users versus nonaspirin users in contrast to the other genotype . All the other PTGS1, PTGS2 and HPGD SNPs did not reach statistical significance for a difference in total colorectal polyp number between aspirin and placebo users according to genotype (Supplementary Table S2). For PTGS1 rs4837960, aspirin use was associated with a reduction in total colorectal polyp risk in homozygotes for the major allele , but not in individuals with one or more minor T alleles , but not common homozygotes .None of the ALOX5 SNP rs7090328 and for three ALOX12 SNPs , for aspirin users . In the negative binomial regression model, ALOX5 rs7090328 was associated with modification of the effect of aspirin, with a reduction in total colorectal polyp risk in homozygotes for the minor allele , but not in individuals with one or more major A alleles , with a Pint value of 0.03 .None of the TP53 tumor suppressor gene . In the negative binomial regression model, colorectal polyp risk reduction by aspirin was significant for rare homozygotes  with a Pint = 0.06, but not for individuals with one or more major alleles .PTGS1), COX-2 (PTGS2), 5-LOX (ALOX5), and 12-LOX (ALOX12) with differential polyp prevention efficacy of aspirin, but not EPA, in the seAFOod trial.Using a panel of SNPs in genes controlling oxylipin signaling relevant to colorectal carcinogenesis and the pharmacology of aspirin and EPA, we report the association of several SNPs in genes encoding COX-1  with another PTGS1 SNP rs3842787, which has been reported to interact with NSAID use for cancer risk  described a possible interaction between rs4648310 and aspirin use (81 mg daily)  and aspirin or EPA use in seAFOod trial participants. rs20417 has been reported to approach statistical significance (P = 0.07) for an interaction with NSAID use for reduction in colorectal adenomatous polyp risk in one case–control study (494 cases and 584 controls), but not in another smaller study . It wilALOX12 SNPs that was associated with differential colorectal polyp prevention activity of aspirin in our study (the intronic SNP rs2920421) has been reported to interact with NSAID use in a case–control study of colorectal cancer risk  database.One of the cer risk . In thatcer risk . In a secer risk . ConsistWe also demonstrated that homozygosity for the SNP  in the coding region of the TP53 tumor suppressor gene was associated with colorectal polyp reduction in aspirin users in the seAFOod trial, in contrast with the group of individuals that had at least one major (G) allele, who did not demonstrate a reduction in colorectal polyp risk associated with aspirin use. At least one C allele at rs104522 has been reported to have an OR of 1.16  for presence of one or more colorectal adenomas compared with homozygous G:G individuals in a meta-analysis of genetic association studies of colorectal adenomas . The rolWe did not observe any gene–supplement interaction for any SNP related to the modest effect of EPA use on colorectal polyp number in the seAFOod trial. The reduction in total colorectal polyp number associated with EPA use in the seAFOod trial was modest and just failed to reach statistical significance . Howevera priori knowledge of aspirin and EPA anticancer pharmacology , if the effect size is only moderate (IRR > 0.6). We also draw attention to the fact that the seAFOod trial population consisted of individuals who had undergone BCSP colonoscopy after a positive FOBt, thereby generating a predominantly male and white ethnicity cohort  interactions from the seAFOod polyp prevention trial. SNPs in COX-1, COX-2, LOX isoforms, and TP53 should be further evaluated as biomarkers of aspirin chemoprevention efficacy, alone and in combination with other polymorphisms reported to predict colorectal polyp and/or colorectal cancer risk reduction by aspirin  colorectal polyp counts according to seAFOod trial treatment allocation by factorial margins and PTGS SNP genotypes.Click here for additional data file.Supplementary Figure 2Supplementary Figure 2 shows the distribution of individual total (combined adenomatous and serrated polyp) colorectal polyp counts according to seAFOod trial treatment allocation by factorial margins and ALOX SNP genotypes.Click here for additional data file.Supplementary Figure 3Supplementary Figure 3 shows the distribution of individual total (combined adenomatous and serrated polyp) colorectal polyp counts according to seAFOod trial treatment allocation by factorial margins and TP53 rs104522 genotype.Click here for additional data file.


Biological collections, including arrayed libraries of single transposon (Tn) or deletion mutants, greatly accelerate the pace of bacterial genetic research. Despite the importance of these resources, few protocols exist for the replication and distribution of these materials. Here, we describe a protocol for creating multiple replicates of an arrayed bacterial Tn library consisting of approximately 6,800 mutants in 96-well plates (73 plates). Our protocol provides multiple checkpoints to guard against contamination and minimize genetic drift caused by freeze/thaw cycles. This approach can also be scaled for arrayed culture collections of various sizes. Overall, this protocol is a valuable resource for other researchers considering the construction and distribution of arrayed culture collection resources for the benefit of the greater scientific community.IMPORTANCE Arrayed mutant collections drive robust genetic screens, but few protocols exist for replication of these resources and subsequent quality control. Increasing the distribution of arrayed biological collections will increase the accessibility and use of these resources. Developing standardized techniques for replication of these resources is essential for ensuring their quality and usefulness to the scientific community. Escherichia coli Keio collection (Bacillus subtilis single-gene knockout libraries (Staphylococcus aureus USA300_FPR3757 transposon (Tn) mutant library  libraries were generated (n = 73 plates), with insertions in approximately 70% of annotated genes and intergenic regions in OG1RF. The second SmarT library consists of 1,946 Tn insertions in poorly characterized genes and intergenic regions and was designed to facilitate genetic screens targeting uncharacterized regions of the genome  to increase the accessibility of this resource for other laboratories, ensure quality control of the collection, and avoid genetic drift by decreasing the number of freeze/thaw cycles for the original library plates. Here, we present a protocol for efficient manual replication of arrayed library resources, including estimation of the time required (person hours). This protocol does not require access to robotic handling systems, making it feasible for researchers who do not have access to this specialized equipment. This protocol can be scaled to accommodate libraries of different sizes, as well as different numbers of replicates. We also describe multiple quality control checks throughout the process and compare sequencing-based verification of pooled mutants with previously published results. Additionally, because of ongoing supply chain difficulties due to the coronavirus disease 2019 (COVID-19) pandemic, we consider multiple options for consumables required throughout, as well as ergonomic considerations for technical staff.We manually created 15 copies of the 73-plate SmarT library over a 2-week period in April 2022. Required supplies and consumables are listed in Multiple quality control checkpoints were used to prevent contamination. The entire process was carried out in a class 2 (A2) biological safety cabinet using biosafety level 2 (BSL-2) practices. Deep-well plates were filled with BHI broth and incubated overnight at 37°C prior to inoculation to ensure a lack of contamination. Replicating pins used for inoculation from freezer stock plates were sterilized in a series of ethanol baths between plates. To ensure against dilution effects that could reduce the effectiveness of alcohol-based disinfection, the ethanol baths were completely replaced after inoculation of four deep-well plates. Replicating pins were also autoclaved every night.600 values and provide these as a data set for the community. Importantly, we did not find any mutants in the entire 6,829-clone library that have lost viability since the initial library construction.The original SmarT library layout included a mapped series of known blank/empty wells in each plate . These wE. faecalis OG1RF (~107 CFU/mL) or medium blanks. The plate was sealed, inverted, and incubated at 37°C overnight. Any well-to-well leakage would result in growth of OG1RF in the medium blank wells. We did not observe any contamination or growth outside the wells that were inoculated  due to the number of samples. To ensure that this would not introduce cross-well contamination, we first empirically investigated the potential for cross-contamination. We filled a 96-well plate with glycerol, buffer, and bromophenol blue for visualization. This plate was covered with a foil seal, vortex-mixed at 2,500 rpm for 5 min, and incubated overnight on a rotating shaker. This agitation exceeded the brief mixing process performed with Tn library plates containing cultures and glycerol. No dye leakage or damage to the foil seal was observed , suggestoculated , strengtin vitro growth defects in liquid medium and to screen each mutant stock for contamination. To determine whether we could pool liquid cultures of the SmarT library after growth in deep-well plates and still achieve a similar balance of mutants, we combined ~200 μL remaining from each deep-well plate after distribution of the cultures into the new library plates. DNA was extracted from the pooled cultures, and Tn abundance was determined using Illumina sequencing and previously established protocols  or similar genetic screens. Previously, the pooled versions were created by plating aliquots of each strain on BHI agar plates, followed by scraping and combining all mutants . This warotocols . These rrotocols .n = 6,829). In the new pooled library, 250 mutants were missing (0 reads) in all extracted replicates  . A total of 192 mutants had 0 reads in all previously sequenced samples  (We first compared the number of Tn mutants identified from sequencing with the known number of mutants in the arrayed library (licates) . A totallicates) . In addilicates) . Low-abulicates) . Howeverlicates) . OverallCulture collections and arrayed mutant libraries are valuable biological resources that increase the throughput, rigor, and reproducibility of experiments across an entire scientific field. To avoid redundancy and wasted resources, these collections and libraries should be broadly available to researchers. While core facilities or private companies may have resources to generate arrayed library copies using robotic arraying and liquid-dispensing equipment, this remains beyond the reach of most academic research laboratories at many institutions. Therefore, we sought to establish a protocol for manual replication of arrayed library collections that would increase the accessibility of these biological resources while maintaining high quality control standards and preventing genetic drift due to repeated freeze/thaw cycles of arrayed culture collections.E. faecalis OG1RF Tn library; we have already distributed most of these library sets to other research groups. We also pooled Tn mutants grown during library replication and used Tn-Seq to compare mutant distribution from this pool to the results for a previously generated pooled library in which individual mutants were scraped from agar plates. Although we found that a majority of Tn mutants (>96%) were present in our new pooled library, we observed greater representation of low-abundance mutants using the previous approach of scraping and pooling mutants from agar plates. The liquid pooling approach described here could have been strengthened by measuring endpoint OD600 values for each library plate after overnight growth and adjusting the volume pooled for each mutant accordingly. Although the plating and scraping approach is more labor-intensive, we strongly recommend using that approach to generate new pooled libraries, instead of pooling together liquid cultures. This methodology can guide the creation and distribution of arrayed mutant collections in a variety of microorganisms.Using this approach, we created 15 copies of a large arrayed E. faecalis arrayed Tn library was previously generated and stored at −80°C  were labeled on the lid and two sides of the plate with printed cryovial labels (LabTAG). Labels contained the library copy number (from 1 to 15) and plate number (from 1 to 73). One hundred microliters of autoclaved 50% glycerol (VWR) was dispensed into each well using multichannel electronic pipettes.From each deep-well plate, 100 μL of overnight culture was distributed into 15 prelabeled 96-well plates (prefilled with 100 μL 50% glycerol) using multichannel electronic pipettes. Sterile AlumaSeal adhesive foil seals (Life Science Co.) were applied with a small rubber roller . Plates were sorted and stored at −80°C.E. faecalis OG1RF (~107 CFU/mL) in BHI broth plus 10% glycerol or medium blanks. The plate was capped with a foil seal, mixed by inversion, and incubated at 37°C overnight, after which an OD600 measurement was taken using a Biotek Synergy H1 plate reader. Data shown are the average of three independent biological replicates.A 96-well plate with 0.1% bromophenol blue in phosphate-buffered saline (pH 8) and 10% glycerol was set up in a checkerboard pattern, with a total volume of 200 μL per well. The plate was capped with an AlumaSeal adhesive foil seal (Life Science Co.) and sealed with a small rubber roller . The plate was mixed by inversion, vortex-mixed for 5 min at 2,500 rpm on a multitube vortex-mixer (Benchmark Scientific), and incubated overnight on a Belly Dancer orbital shaker at ~50 rpm. Leakage of dye between wells was evaluated by eye. For quantitative measurement of intraplate leakage, a 96-well plate was inoculated with alternating wells of Two hundred microliters was pooled from each well of each deep-well plate after the distribution of mutants to new library plates. Samples were pooled in 50-mL Falcon tubes, pelleted at 6,000 rpm for 10 min in a Beckman Coulter Avanti JXN-30 floor centrifuge, and stored at −20°C until further use. DNA was extracted using a Qiagen DNeasy blood and tissue kit with a lysozyme pretreatment step, as described previously , and subGSE233193.Tn-Seq data have been deposited in the NCBI GEO database under accession number


We created five datasets to train two CNNs, LeNet and AlexNet: (1) 128FOV used a 0° projection image without preprocessing, (2) 40FOV used 0° projection images cropped to 40 × 40 pixels centered on the striatum, (3) 40FOV training data doubled by data augmentation , (4) 40FOVhalf, and (5) 40FOV_DAhalf, split into left and right (20 × 40) images of 40FOV and 40FOV_DA to separately evaluate the left and right SBR. The accuracy of the SBR estimation was assessed using the mean absolute error, root mean squared error, correlation coefficient, and slope. The 128FOV dataset had significantly larger absolute errors compared to all other datasets (p < 0. 05). The best correlation coefficient between the SBRs using SPECT images and those estimated from frontal projection images alone was 0.87. Clinical use of the new CNN method in this study was feasible for estimating the SBR with a small error rate using only the frontal projection images collected in a short time.This study aimed to develop a new convolutional neural network (CNN) method for estimating the specific binding ratio (SBR) from only frontal projection images in single-photon emission-computed tomography using [ Employing [123I]-FP-CIT SPECT increases the precision of choosing the best treatment options and aids in the early detection of Parkinsonian syndrome and dementia with Lewy bodies [123I]-FP-CIT SPECT. Currently, in clinical practice, quantitative evaluation by SBR plays an important role as an adjunct to visual evaluation.Single-photon emission-computed tomography (SPECT) can show the distribution of the dopamine transporter (DAT) in the brain using 23I iofluy bodies ; howevery bodies ,6,7. The123I]-FP-CIT SPECT exams are often conducted, and clinical practice guidelines have been published in various regions [123I]-FP-CIT SPECT scans in Japan are performed in accordance with the ‘Clinical Ioflupane Guidelines’ of the Japanese Society of Nuclear Medicine, available online at http://www.jsnm.org/archives/1151/ (accessed on 6 April 2023). To get sufficient acquisition counts, these guidelines indicate an appropriate acquisition time of 30 to 45 min. Short-time scans reduce the acquisition counts, deteriorate the quality of the visual evaluation, and underestimate SBR [123I]-FP-CIT SPECT is important to evaluate striatal functions, some patients cannot tolerate the static position for reasons described above. If a frontal projection image with only a short time duration can estimate striatal functions in such cases, the image analysis method may provide appropriate clinical diagnosis.Worldwide, -FP-CIT SPECT, and it also aimed to evaluate the error and correlation between SBR estimated by the new CNN method and SBR obtained by the full SPECT study. If patients who cannot undergo the exam for the required amount of time could receive an accurate estimate of SBR from a rapid frontal projection, the results of our investigation would be of great clinical value. Nakajima et al. developed a machine learning (ML) method that could diagnose Parkinsonian syndrome and dementia with Lewy bodies [Our study aimed to develop a new CNN method for estimating SBR from only frontal projection images in 3I-FP-CIT123I]-FP-CIT SPECT at the University of Fukui Hospital from January 2017 to December 2021 were included in this retrospective study. The Ethics Committee of our university’s Faculty of Medical Sciences approved the study’s protocol (#20210156) and waived the requirement of obtaining patient informed consent and approved a retrospective evaluation of imaging and clinical data. Patients received a gradual intravenous infusion of approximately 170 MBq [123I]-FP-CIT in the morning, and 3–4 h later, they had SPECT scans (167 MBq at noon).Four hundred twenty patients (72.1 ± 9.7 y) who underwent -FP-CIT uptake in the parotid gland. In the Grad-CAM of the 128FOV  had larger errors than the 40FOV and 40FOV_DA, it demonstrated a strong correlation between the SBRs with full SPECT data and the SBRDL. Bland–Altman plots revealed that the 128FOV dataset had a larger systematic error than the 40FOV and 40FOV_DA datasets. This suggests that the increase in cases did not improve the SBR estimation accuracy in the 128FOV dataset. Therefore, 128FOV projection data alone (image without trimming processing) are inappropriate for SBR estimation. It seems that noise in the large space outside the head may have affected this result because 40FOV shows a slightly similar trend. These results showed the same tendency in both the DaT View and the count-based methods. We consider calculating the left and right SBR independently to be clinically valuable since patients with Parkinsonian syndrome often have left–right differences in SBR -FP-CIT SPECT estimated the SBR with high correlation and low errors by using a preprocessed dataset for the projection image. The estimation accuracy was improved by increasing the training cases through data augmentation. Clinical use of the new CNN method in this study is considered feasible since SBR can be estimated with a small error rate using only frontal projection images collected in a short time.The 0° projection image in [


Abyssal seafloor communities cover more than 60% of Earth’s surface. Despite their great size, abyssal plains extend across modest environmental gradients compared to other marine ecosystems. However, little is known about the patterns and processes regulating biodiversity or potentially delimiting biogeographical boundaries at regional scales in the abyss. Improved macroecological understanding of remote abyssal environments is urgent as threats of widespread anthropogenic disturbance grow in the deep ocean. Here, we use a new, basin-scale dataset to show the existence of clear regional zonation in abyssal communities across the 5,000 km span of the Clarion–Clipperton Zone (northeast Pacific), an area targeted for deep-sea mining. We found two pronounced biogeographic provinces, deep and shallow-abyssal, separated by a transition zone between 4,300 and 4,800 m depth. Surprisingly, species richness was maintained across this boundary by phylum-level taxonomic replacements. These regional transitions are probably related to calcium carbonate saturation boundaries as taxa dependent on calcium carbonate structures, such as shelled molluscs, appear restricted to the shallower province. Our results suggest geochemical and climatic forcing on distributions of abyssal populations over large spatial scales and provide a potential paradigm for deep-sea macroecology, opening a new basis for regional-scale biodiversity research and conservation strategies in Earth’s largest biome. Analysing seabed imagery data spanning 5,000 km and >50,000 megafaunal specimens across the Clarion–Clipperton Zone abyssal plain in the northeast Pacific, the authors identify two distinct biogeographical provinces in which taxa that are dependent on calcium carbonate structures are restricted to depths above 4,300–4,880 m. This abyssal seabed consists of plains and hills that extend across ocean basins, interspersed with seamounts and subdivided by mid-ocean ridges, ocean trenches and fracture zones1. Lacking sunlight, energy in this environment is highly limited, with detrital particles sinking from surface waters providing the main source of food2, while temperatures are steady at 0.5–3.0 °C (ref. 3) and bottom currents are generally low (0–0.25 m s−1) (ref. 4). As a result, abyssal benthic communities typically exhibit low abundance and biomass compared with other marine environments5 but they support high species richness at regional to landscape scales7 and play an active role in the cycling of carbon8. Abyssal habitats are therefore considered to be reservoirs for biodiversity9 and sources of important ecosystem services10. Despite their remoteness and as a consequence of being energy-restricted and environmentally stable, abyssal habitats are also expected to be highly vulnerable to anthropogenic disturbances including climate change12 and emerging industrial activities such as polymetallic nodule mining14. To adequately conserve the biodiversity and services of abyssal seabed ecosystems, it is critical to consider and protect their full range of habitats and communities, which requires elucidating the patterns and processes controlling the distribution of abyssal populations over large, regional scales.The abyssal seabed lies between water depths of 3,000 and 6,000 m  bathyal seafloor ecosystems22, which encompass a variety of relatively well-known habitats, between 1,000 and 3,000 m depth, including continental slopes, submarine canyons and coral mounds. Consequently, while biogeographic provinces proposed for the deep ocean readily distinguish bathyal and abyssal provinces within oceanic basins24, potential variation within abyssal regions remains largely unresolved a decade after provincial boundaries based on surrogate environmental data were postulated24, since large-scale empirical studies  are lacking for remote abyssal ecosystems. As such, the processes regulating alpha- and beta-diversity across the scale of a seemingly connected abyssal plain remain unclear, particularly for the largest size-class of seafloor organisms, megafauna . Megafauna are a conspicuous component of the abyssal benthos and a common target for investigation in deep-sea spatial ecology27 because occurrences of these taxa can be determined across large spatial scales from seabed imagery28 collected by deep-sea robots29.Macroecological studies assessing spatial variation on regional to global scales in the deep ocean2 of abyssal plain in areas beyond national jurisdiction between the Exclusive Economic Zones of Kiribati and Mexico . Our dataset spans 5,000 km across the CCZ  encompassing >50,000 megafaunal specimens from 411 morphotypes  in 13 phyla, enabling investigation of ecological gradients  in seabed communities across the region to develop key understanding of macroecological patterns in Earth’s largest biome.To address these knowledge gaps, we compiled a new basin-scale standardized dataset to investigate patterns of abundance and diversity in benthic megafaunal communities across the Clarion–Clipperton Zone . The CCZ spans ~6 million kmico Fig.  and is t−1) but decreased in abundance by over an order of magnitude below 4,300 m (mean density ~100 ind ha−1), becoming virtually absent below 4,800 m (mean density <10 ind ha−1); (2) brittle stars (Ophiuroidea) were most abundant at shallow-abyssal depths , numerically dominated assemblages at intermediate depths  but decreased to low abundances below 4,800 m (mean density <40 ind ha−1); (3) anemones (Actiniaria) consistently increased in relative abundance with depth, first replacing soft corals as the dominant Cnidaria group below 4,300 m and then replacing brittle stars as the most dominant group in assemblages below 4,800 m , were absent below 4,400 m, where cirrate octopuses  became the only molluscan group in megabenthic assemblages. Changes in dominant taxa at the class or phylum level  sea0 m Fig. ; (5) shevel Fig.  clearly vel Fig. : a shallvel Fig. .Fig. 2VaOphiosphalma glabrum  and Thenea sp. indet.  were found exclusively in this deep zone, while 64 morphotypes (45 rare) out of a total of 283 were found exclusively in the shallow province. However, only 25 morphotypes (23 rare) from a total of 264 were found exclusively in the transitional zone. From the total of 175 (14 rare) morphotypes that were found in both shallow and deep communities, only two were within the ten most dominant taxa in both the shallow province (first- and third-most abundant) and in the deep province . Below the CCD, calcium carbonate (CaCO3) is undersaturated, which can energetically constrain the development and distribution of species highly dependent on carbonate structures such as in calcareous Foraminifera, a ubiquitous protozoan group showing clear depth zonation at intermediate scales in the abyss38. It is these groups, probably depending on CaCO3 structures, that exhibit the greatest variation in relative abundance or even presence with depth in our analysis and thereby contribute most to regional beta-diversity. For example, shelled molluscs  were absent below 4,400 m and soft corals (with scleroblast cells requiring CaCO3 to produce sclerites40) and bryozoans (most species dependent on CaCO3 exoskeletons41) were replaced by ‘soft’ anemones (Actiniaria) below 4,300 m. The echinoderm transition from ophiuroids to holothurians below 4,800 m, yielding the dominant taxon of the deep province, could be related to either differing CaCO3 requirements or metabolic efficiency, documented across echinoderms42. All of these shifts were observed within the depth range  of the CCD in the northeast Pacific36. However, our understanding of biogeochemical and metabolic cycles in abyssal ecosystems is still too limited to fully comprehend the specific role of the CCD and calcium calcite saturation, as a biogeographical driver in abyssal metazoans.The nature of the taxonomic shifts between provinces and the depth range at which these occur suggests that the carbonate compensation depth (CCD) plays a key role in the depth zonation we encountered. The CCD lies close to the seafloor depth in the northeast Pacificq = 0) ranged between 32 and 62 morphotypes per 200 specimens in the shallow-abyssal province and between 48 and 63 in the deep one but we found no evidence of a difference in richness across the three depth ranges  and Shannon diversity  with increasing depth  was up to an order of magnitude larger in the shallow than in the deep province , increasing towards southeast locations  in closer proximity to the high primary productivity related to equatorial upwelling8, with known capacity to regulate abyssal benthic processes46. Similarly, the benthic communities surveyed in northeasternmost locations —for instance, with the largest densities we observed in scavenging megafauna  where a previous top-down environmental assessment identified the potential for two abyssal provincesces Fig. , with mence Fig. . Nonethence Fig.  than depnce Fig. , especiance Fig. . In partnce Fig.  probably22, a reduction that is thought to reflect the wider geographic ranges of deep-sea species23. But our results suggest that more complex processes may control diversity in the abyss, helping maintain community richness with depth. These interpretations could be affected by the historical unbalance in the sampling conducted in abyssal ecosystems  compared to shallower deep-sea environments52. In turn, our results add to the existing evidence53 against a source–sink dependency from bathyal to abyssal communities in the Pacific54 and stress that, given the wealth of between-phylum species replacement we encountered with increasing depth, assessments including multiple phyla might be best suited to investigate biodiversity and macroecological patterns in abyssal regions.The results of this study represent the largest multiphylum assessment of beta-diversity patterns to date conducted across such a vast abyssal seabed extension , provide key insights to our view of abyssal ecosystems and biodiversity and challenge some current paradigms in deep-sea macroecology. The maintenance of high taxonomic richness with increasing depth by phylum-level replacements (in presence or dominance) was surprising. Diversity, especially that of the largest-sized fauna, is commonly expected to decrease with depth and nutrient flux in the deep sea38), although more physiological research is needed to better understand how CaCO3 saturation affects different phyla. Greater understanding of CaCO3 saturation as a biodiversity driver is especially important as the CCD is expected to shoal with increased CO2 levels in the ocean associated with climate change55. In the light of our results, shoaling of the CCD could have much larger implications for abyssal biodiversity than previously expected, especially in regions like the northeast Pacific where the seafloor depths transcend the current CCD11. A CCD shoaling of tens of metres in abyssal ecosystems, given their vast area, could lead to shifts in the environmental conditions  across thousands of km2 of seabed worldwide. This could trigger large shifts in community structure and species distributions or even extinctions of highly specialized abyssal taxa, adding to the cumulative impacts of deep-sea mining and other emerging anthropogenic disturbances56 that might concur on Earth’s largest biome in the coming years.The presence of such a clear and unexpected boundary zone demarking the limits of provinces has not been previously documented in abyssal metazoans, opening questions for further macroecological research in the deep ocean. We suggest that the CCD may control the location of the environmental boundaries between shallow and deep-abyssal provinces for megafauna, as appears to occur in protozoans  lies close to the seafloor in the northeast Pacific35, deepening north to south between 4,400 and 4,800 m (ref. 36). Surface sediments in the northeast Pacific are relatively well-oxygenated and generally dominated by either siliciclastic clay and radiolarian ooze in the northern and central parts of the CCZ, or by biogenic calcareous oozes and fine-grained sediments in southern sites45. These are supplied by extremely low sedimentation rates, regionally varying between 0.2 and 1.15 cm per 1,000 years61. These conditions are thought to promote the (extremely slow) formation of polymetallic nodules35, yielding growth rates of ∼1–12 mm per million years in the abyssal Pacific31. Nodules can vary in size, shape and abundance61 and are extensively distributed across the northeast Pacific31 but are not exclusively found there. Vast abyssal nodule field areas are present in the southeast Pacific , the southwest Pacific  and in the Central Indian Ocean Basin31. Nodule fields form an unusual mosaic habitat, where the hard substratum provided by nodules acts as keystone structure increasing local seabed complexity63, promoting the occurrence of some of some of the most biodiverse benthic assemblages surveyed in the abyss27. But the biodiversity of the CCZ is poorly known, for example 90% of the species identified in the region are thought to be new to science51 and substantial levels of dark biodiversity have been predicted34. In addition, a general lack of standardization of intercalibrated taxonomic standards for the identification of benthic fauna50 has historically hampered comparability between biological studies64 and thereby the assessment of regional, macroecological patterns across the northeast Pacific basin.The CCZ is an extensive abyssal plain and hill ecosystem, interspersed by seamount areaswaters59 . In turn2; (4) no overlapping frames included in image-based studies; (5) scalable stills collected vertically facing the seabed . Imagery data were collected within 14 sites managed by the International Seabed Authority across the CCZ , and by reference to existing literature69—though developed before the major higher taxonomic revision recently undergone by Octocorallia70. The catalogue follows the ref. 33 open nomenclature to report the taxonomic resolution reached in each morphotype but all taxa identified from the catalogue (411 morphotypes in this study) were deemed as sufficiently different morphologically by taxonomic experts to be confidently considered separate species. Specimens with uncertain classification at the morphotype level  but identifiable to a certain higher taxonomical level (like phylum or class) were retained only for density analyses. Taxa living in a closed shell or tube  were excluded from analysis as it is not possible to determine whether these are alive in images. Several spatially blind reviews were conducted to the whole dataset to ensure a robust taxonomic alignment between sites, consisting in side-by side visualization of all specimens classified under the same catalogue label using the ‘Label Review Grid Overview’ tool in BIIGLE65; a process that was repeated many times by the same group of expert seabed image scientists, to minimize potential bias28. In addition, the likely feeding behaviour  of each morphotype was inferred from observational knowledge and by reference to similar organisms described in the literature. With a total of 53,512 megafaunal specimens in 13 phyla, our biodiversity dataset was compiled across a wide geographical span  at the abyss and is of comparable magnitude to the largest datasets that have been used to underpin our theoretical understanding in the deep-sea .Megafauna specimens >10 mm were detected and counted in imagery reanalysed from the selected surveys  within sites. This process divided the SSdat into 23 locations and the BDdat into 28 locations . In locations where surveys covered a large depth spectrum, data were further constrained to 200 m depth bins during replicate sample generation. Following recommendations of minimum sample size for accurate and precise estimation of local megabenthic community features47 and to minimize the potential effect of regional gradients in faunal abundance on diversity measures , we generated SSdat samples containing 450–500 specimens  and BDdat samples containing an exact number of 200 specimens (taxonomic resolution including only specimens identified to morphotype level). This process yielded a total of 84 samples standardized for density analyses (standing stocks) and 161 samples standardized for biodiversity analyses (richness and beta-diversity).We compiled invertebrate megafauna occurrences into two different subsets to investigate variations across space in different parameters. These were 1) standing stocks subset (SSdat): containing faunal count data at all taxonomical levels obtained only from scalable still images and thereby associated with a precise measure of the seabed surface area ; and (2) biodiversity subset (BDdat): containing faunal count data obtained from both image and video surveys but including only specimens identified up to morphotype level . Using ArcMap v.6.10 software, we interpolated each specimen across a 10 × 10 km standingorg m−2 yr−1) to the seabed derived by applying vertical flux attenuation59 to satellite-derived export flux data (2) of seabed surveyed in each SSdat sample; that is, for the whole assemblage, specific populations or faunal groups of interest  and (2) to examine the range of diversity characteristics, Hill’s diversity numbers of order 0 and 1 (ref. 43) were calculated in each BDdat sample as morphotype richness (S), the exponential form of the Shannon diversity index (expH’), for the whole assemblage.Depth and position (latitude and longitude) of specimens (or images) were averaged within each sample to investigate variations in communities across space and environmental gradients. Sample positions were interpolated with predictions of yearly POC flux  and two main environmental factors .Preliminary exploration of the interaction between different environmental factors  across study locations revealed substantial correlations between depth and longitude and also between POC flux and latitude .We followed a simple, data-driven, bottom-up approach to investigate the potential existence of biogeographic boundaries across the depth spectrum of the north Pacific abyss. First, using the whole dataset, we generated ridgeline plots depicting within-group geometric distributions of the total abundance sampled in the most dominant high taxonomic groups . On the basis of the results of this analysis, we assessed the potential variability within and between the megabenthic communities in tentative biogeographical provinces, by grouping samples in three different depth bins: shallow <4,300 m; transition 4,300–4,800 m; deep >4,800 m). Mean values and 95% confidence intervals were calculated for different ecological parameters  within each tentative province. To contextualize patterns in morphotype richness and the representability of the sampling conducted within each depth range, morphotype accumulation curves were calculated following ref. 77) taxon abundances. Non-metric multidimensional scaling ordinations were conducted to visualize the rate of dissimilarity (distance) between all pairs of samples; isotropic contour lines depicting broadly approximated depth ranges were fitted using generalized additive models78 in the resulting multidimensional scaling plots to aid visualization of beta-diversity patterns across environmental gradients. All data processing and analysis were implemented in R v.4.2.1 (ref. 79), using standard analysis methods and functions from the packages: ‘vegan’80, ‘AICcmodavg’81, ‘ggridges’82 and ‘ggplots2’83. We report statistical evidence of variations in ecological parameters across environmental gradients  or between biogeographical provinces  using the simplified language of evidence84.Variations in community composition across locations, sites and potential biogeographical provinces were further explored using BDdat samples. Dissimilarity in faunal composition between all pairs of samples was calculated using the Bray–Curtis dissimilarity measure on square-root transformed (normalizedFurther information on research design is available in the Supplementary InformationSupplementary Methods, Figs. 1–5 and Tables 1 and 2.Reporting Summary


Growing concern around global warming has led to an increase in research focused on plant responses to increased temperature. In this review, we highlight recent advances in our understanding of plant adaptation to high ambient temperature and heat stress, emphasizing the roles of plant light signaling in these responses. We summarize how high temperatures regulate plant cotyledon expansion and shoot and root elongation and explain how plants use light signaling to combat severe heat stress. Finally, we discuss several future avenues for this research and identify various unresolved questions within this field. Most people living in Europe, China, and the US have experienced extreme heat waves over the course of this summer Witze, . ClimateArabidopsis thaliana as an example. In these plants, increases in temperature from their normal growth (22 °C) to high ambient temperature (28 °C) induce a series of morphological changes (thermomorphogenesis), including hypocotyl elongation, leaf petiole elongation, root elongation, reduced cotyledon expansion, and leaf hyponasty  and heat stress temperatures are 37–45 °C.Over the past decade, many well-designed studies have illustrated how plants sense and signal high temperatures to regulate their growth and development. Interestingly, plant photoreceptors and light-signaling components play crucial roles in plant thermomorphogenesis, with several elegant review papers summarizing the interactions between light and temperature  senses short-wavelength UV-B light (280–320 nm) . Unlike other photoreceptors, phytochromes are found in both active and inactive forms that are reversibly switched under red or far-red light irradiation. In these systems, red light absorption activates the phytochromes (Pfr form) and induces their translocation to the nucleus, where they trigger the red light response. However, far-red light treatment inactivates Pfr, returning them to their inactive form and inhibiting their signaling function  acting as the central negative regulator of these systems. COP1, initially identified using forward genetic screening in the 1990s  transcription factor family. These seven PIF proteins play partially redundant roles, although several are also known to perform fairly specific functions under certain conditions  known to be largely insensitive to high temperatures at both the phenotype and genome-wide transcriptome levels  results in a significant reduction in PIF4 expression. This data also revealed that spaQ plants presented with much shorter hypocotyls under high ambient temperature conditions  has been identified as a potent regulator of cotyledon expansion, with spt mutants displaying significantly expanded cotyledons with larger pavement cells than the wild-type plants when grown under red-light conditions , to increase cotyledon endoreplication and cell size   into indole-3-acetic acid (IAA)  to the YUCCA8 promoter . Although EIN3 does not directly interact with PIF4, EIN3 does activate the expression of PP2C.D , repressing H+-ATPase activity  and pifQ (pif1 pif3 pif4 pif5) quadruple mutants shown to be similar to those of wild-type plants  to restrict HY5 protein movement from shoot to root, results in the hy5 mutant background (pCAB3:DOF-HY5/hy5) not being able to rescue the hy5 short root phenotypes under high ambient temperature  involved in brassinosteroid metabolism, allowing this factor to modulate brassinosteroid signaling  in the hy5 background mediated clear root elongation . However, when plants grow in temperatures above this limit, they no longer experience thermomorphogenesis but rather enter heat stress. Heat stress has many adverse effects on plant growth and development, including inhibition of germination, growth, reduced fertility, and cell death (heat stress responses)  , inducing their transcription and supporting the transition to senescence in response to heat treatment  growth conditions reveal a clear increase in heat shock (45 °C for 45 min) tolerance in the latter group  acts as the key transcription factor in the abscisic acid (ABA) signaling pathway, which, in turn, regulates seed germination. Heat promotes COP1 movement from the nucleus to the cytoplasm, increasing HY5 stability and promoting its interactions with the Taken together, these results show that light signaling components are also critical to the heat stress response in plants Fig. .Fig. 2RoEvaluation of the precise temperature sensing mechanisms. This is because plants tend to reduce growth in response to lower temperatures following chilling or freezing stress, albeit to different degrees, but increase their growth in response to higher ambient temperatures , eventually entering restricted growth when temperatures reach the high-temperature stress threshold (Phase 2). We do not know how a plant measures these temperature changes or makes contrasting decisions to enter either of these two phases. Previous studies have focused only on phase 1 or phase 2, and further studies need to focus on their cross-over and simultaneous induction, with initial investigations focusing on known photoreceptors, including PhyB.The roles of PIF4 in different developmental stage and temperature range are deserved to be further investigated. As we described, PIF4 positively regulates cell elongation in hypocotyl cells but negatively controls cell expansion in cotyledon cells under high ambient temperatures. Moreover, PIF4 enhances plant heat stress tolerance in seedlings but triggers senescence in adult plants. Therefore, we assume that the function of PIF4 varies according to distinct stages. The recently popular single cell analysis may contribute to a detailed understanding of PIF4 in a more spatial and temporal way.The mechanisms underlying plant root thermomorphogenesis are not well understood. Although HY5-mediated signaling is required for root elongation at high ambient temperatures, other mechanisms are worthy of further study. This is important because we know that detached roots can elongate under high ambient temperatures, even in the absence of cotyledons and hypocotyls (Bellstaedt et al., 6A writer proteins to regulate N6-methyladenosine modification and circadian rhythm (Wang et al., The LLPS of thermosensors, phyB and ELF3, suggests that high temperature-induced condensates are a common regulatory mechanism in plants, facilitating their nuanced response to changes in temperature. Since CRY2 also undergoes LLPS in response to blue light and co-condense with mFinally, although we clearly understand these processes in Arabidopsis, there is an urgent need to evaluate its translational value in other plants to transfer this knowledge to the cultivation of food and fruit crops and increase their high-temperature tolerance.Although we have achieved many milestones in understanding the high-temperature signaling response in plants, several unexplored phenomena need to be studied. These include: