Search

Capillary pericytes are important regulators of cerebral blood flow, blood-brain barrier integrity and neuroinflammation, but can become lost or dysfunctional in disease. The consequences of pericyte loss or dysfunction is extremely difficult to discern when it forms one component of a complex disease process. To evaluate this directly, we examined the effect of adult pericyte loss on mouse voluntary movement and motor function, and physiological responses such as hypoxia, blood-brain barrier (BBB) integrity and glial reactivity. Tamoxifen delivery to Pdgfrβ-CreER T2 :: Rosa26-DTA transgenic mice was titrated to produce a dose-dependent ablation of pericytes in vivo. 100mg/kg of tamoxifen ablated approximately half of all brain pericytes, while two consecutive daily doses of 300mg/kg tamoxifen ablated >80% of brain pericytes. In the open field test, mice with ∼50% pericyte loss spent more time immobile and travelled half the distance of control mice. Mice with >80% pericyte ablation also slipped more frequently while performing the beam walk task. Our histopathological analyses of the brain revealed that blood vessel density was unchanged, but vessel lumen width was increased. Pericyte-ablated mice also exhibited: mild BBB disruption; increased neuronal hypoxia; astrogliosis and increased IBA1 + immunoreactivity, suggestive of microgliosis and/or macrophage infiltration. Our results highlight the importance of pericytes in the brain, as pericyte loss can directly compromise brain health and induce behavioural alterations in mice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Pathologic antibody mediated rejection (pAMR) evaluation and donor specific antibody (DSA) testing are recommended in the first year after heart transplantation (HTx) in adult patients. Whether DSA testing adds prognostic information to contemporary pAMR surveillance has not been fully studied., Methods: This was a single center study of consecutive endomyocardial biopsies (EMB) performed between November 2010 and February 2023 in adult HTx patients. The primary objective was to evaluate whether DSA testing contributes additional information to pAMR surveillance to better predict overall survival. Secondary endpoints included cardiac allograft dysfunction and loss., Results: A total of 6,033 EMBs from 544 HTx patients were reviewed for the study. The pAMR+/DSA+ group had significantly lower overall survival versus the pAMR-/DSA- group (hazard ratio [HR] = 2.63; 95% confidence interval [CI], 1.35-5.11; p c = 0.013). In the pAMR+/DSA+ group, patients with cardiac allograft dysfunction, compared to those without allograft dysfunction, had significantly lower overall and cardiac survival (p c < 0.001 for both). In contrast, pAMR+/DSA+ and pAMR-/DSA- patients without cardiac allograft dysfunction showed no difference in overall and cardiac survival. Primary graft dysfunction (PGD) was a novel risk factor for development of de novo DSAs (dnDSA) three weeks post-HTx (p = 0.007)., Conclusions: DSA testing as the primary surveillance method can identify high-risk pAMR+/DSA+ patients. Surveillance pAMR testing in the contemporary era may need to be reevaluated. Earlier DSA testing at 10-14 days post-HTx should be considered in PGD patients.

The contraction and subsequent death of brain pericytes may play a role in microvascular no-reflow following the reopening of an occluded artery during ischemic stroke. Mammalian target of rapamycin (mTOR) inhibition has been shown to reduce motility/contractility of various cancer cell lines and reduce neuronal cell death in stroke. However, the effects of mTOR inhibition on brain pericyte contraction and death during ischemia have not yet been investigated. Cultured pericytes exposed to simulated ischemia for 12 h in vitro contracted after less than 1 h, which was about 7 h prior to cell death. Rapamycin significantly reduced the rate of pericyte contraction during ischemia; however, it did not have a significant effect on pericyte viability at any time point. Rapamycin appeared to reduce pericyte contraction through a mechanism that is independent of changes in intracellular calcium. Using a mouse model of middle cerebral artery occlusion, we showed that rapamycin significantly increased the diameter of capillaries underneath pericytes and increased the number of open capillaries 30 min following recanalisation. Our findings suggest that rapamycin may be a useful adjuvant therapeutic to reduce pericyte contraction and improve cerebral reperfusion post-stroke., (© 2024. The Author(s).)

Mechanistic understanding of the immune checkpoint receptor PD1 is largely based on mouse models, but human and mouse PD1 orthologs exhibit only 59.6% identity in amino acid sequences. Here we show that human PD1 is more inhibitory than mouse PD1 due to stronger interactions with the ligands PDL1 and PDL2 and with the effector phosphatase Shp2. A novel motif highly conserved among PD1 orthologs in vertebrates except in rodents is primarily responsible for the differential Shp2 recruitment. Evolutionary analysis suggested that rodent PD1 orthologs uniquely underwent functional relaxation, particularly during the K-Pg boundary. Humanization of the PD1 intracellular domain disrupted the anti-tumor activity of mouse T cells while increasing the magnitude of anti-PD1 response. Together, our study uncovers species-specific features of the PD1 pathway, with implications to PD1 evolution and differential anti-PD(L)1 responses in mouse models and human patients.

The close spatial relationship between microglia and cerebral blood vessels implicates microglia in vascular development, homeostasis and disease. In this study we used the publicly available Cortical MM^3 electron microscopy dataset to systematically investigate microglial interactions with the vasculature. Our analysis revealed that approximately 20% of microglia formed direct contacts with blood vessels through gaps between adjacent astrocyte endfeet. We termed these contact points "plugs". Plug-forming microglia exhibited closer proximity to blood vessels than non-plug forming microglia and formed multiple plugs, predominantly near the soma, ranging in surface area from ∼0.01 μm 2 to ∼15 μm 2 . Plugs were enriched at the venule end of the vascular tree and displayed a preference for contacting endothelial cells over pericytes at a ratio of 3:1. In summary, we provide novel insights into the ultrastructural relationship between microglia and the vasculature, laying a foundation for understanding how these contacts contribute to the functional cross-talk between microglia and cells of the vasculature in health and disease., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: HLA-DQ mismatch has been identified as a predictor of de novo donor-specific HLA antibody formation and antibody-mediated rejection. There are insufficient data to guide the incorporation of DQ mismatch into organ allocation decisions., Methods: We used a retrospective longitudinal cohort of adult living donor kidney transplant recipients from 11 centers across the United States for whom high-resolution class II typing was available. HLA-DQαβ heterodimer allele mismatch was quantified for all donor-recipient pairs, and outcome data were obtained through linkage with the Scientific Registry of Transplant Recipients., Results: We studied 3916 donor-recipient pairs. Recipient characteristics were notable for a median age of 51 (38-61) y, primarily unsensitized, with 74.5% of the cohort having 0% calculated panel-reactive antibody, and 60.4% with private insurance, for a median follow-up time of 5.86 y. We found that the HLA-DQαβ allele and HLA-DR antigen mismatch were each individually associated with an increased hazard of all-cause graft failure (adjusted hazard ratio [aHR] DQ = 1.03 1.14 1.28; aHR DR = 1.03 1.15 1.328), death-censored graft failure (aHR DQ =1.01 1.19 1.40; aHR DR = 0.099 1.18 1.39), and rejection. Having 2 HLA-DQαβ allele mismatches further increased the hazard of rejection even when controlling for HLA-DR mismatch (aHR 1.03 1.68 2.74)., Conclusions: HLA-DQαβ allele mismatch predicted allograft rejection even when controlling for HLA-DR antigen mismatch and were both independently associated with increased risk of graft failure or rejection in adult living kidney transplant recipients. Given the strong burden of disease arising from the HLA-DQ antibody formation, we suggest that HLA-DQαβ should be prioritized over HLA-DR in donor selection., Competing Interests: G.P.M. received research support from Thermo Fisher/One Lambda, CareDx, and PIRCHE AG, as well as travel support to scientific meetings from Thermo Fisher. The other authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Regulatory T (Treg) cells are essential for immune tolerance of embryo implantation, and insufficient Treg cells are implicated in early pregnancy loss. An abortion-prone mouse model was used to evaluate the utility of IL-2 complexed with JES6-1 anti-IL-2 antibody (IL-2/JES6-1) to boost uterine Treg cells and improve reproductive success. IL-2/JES6-1, but not IL-2/IgG control, administered in the periconception phase to CBA/J females mated with DBA/2 males elicited a greater than twofold increase in the proportion of CD4 + T cells expressing forkhead box P3 (FOXP3), and an increase in the ratio of FOXP3 + Treg cells/FOXP3 - T conventional cells, in the uterus and its draining lymph nodes at embryo implantation that was sustained into midgestation. An attenuated phenotype was evident in both thymic-derived and peripheral Treg cells with elevated cytotoxic T-lymphocyte antigen-4, CD25, and FOXP3, indicating improved suppressive function, as well as increased proliferative marker Ki-67. IL-2/JES6-1 treatment reduced fetal loss from 31% to 10%, but this was accompanied by a 6% reduction in late gestation fetal weight, despite comparable placental size and architecture. Similar effects of IL-2/JES6-1 on Treg cells and fetal growth were seen in CBA/J females with healthy pregnancies sired by BALB/c males. These findings show that expanding the uterine Treg cell pool through targeting IL-2 signaling is a strategy worthy of further investigation for mitigating immune-mediated fetal loss., Competing Interests: Disclosure Statement None declared., (Copyright © 2024. Published by Elsevier Inc.)

Durable host plant resistance (HPR) to insect pests is critical for sustainable agriculture. Natural variation exists for aphid HPR in sorghum (Sorghum bicolor), but the genetic architecture and phenotype have not been clarified and characterized for most sources. In order to assess the current threat of a sorghum aphid (Melanaphis sorghi) biotype shift, we characterized the phenotype of Resistance to Melanaphis sorghi 1 (RMES1) and additional HPR architecture in globally admixed populations selected under severe sorghum aphid infestation in Haiti. We found RMES1 reduces sorghum aphid fecundity but not bird cherry-oat aphid (Rhopalosiphum padi) fecundity, suggesting a discriminant HPR response typical of gene-for-gene interaction. A second resistant gene, Resistance to Melanaphis sorghi 2 (RMES2), was more frequent than RMES1 resistant alleles in landraces and historic breeding lines. RMES2 contributes early and mid-season aphid resistance in a segregating F 2 population; however, RMES1 was only significant with mid-season fitness. In a fixed population with high sorghum aphid resistance, RMES1 and RMES2 were selected for demonstrating a lack of severe antagonistic pleiotropy. Associations with resistance colocated with cyanogenic glucoside biosynthesis genes support additional HPR sources. Globally, therefore, an HPR source vulnerable to biotype shift via selection pressure (RMES1) is bolstered by a second common source of resistance in breeding programs (RMES2), which may be staving off a biotype shift and is critical for sustainable sorghum production., (© 2024 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

HLA matching in solid organ transplant is performed with the aim of assessing immunologic compatibility in order to avoid hyperacute rejection and assess the risk of future rejection events. Molecular mismatch algorithms are intended to improve granularity in histocompatibility assessment and risk stratification. PIRCHE-II uses HLA genotyping to predict indirectly presented mismatched donor HLA peptides, though most clinical validation studies rely on imputing high resolution (HR) genotypes from low resolution (LR) typing data. We hypothesized that use of bona fide HR typing could overcome limitations in imputation, improving accuracy and predictive ability for donor-specific antibody development and acute rejection. We performed a retrospective analysis of adult and pediatric kidney transplant donor/recipient pairs (N = 419) with HR typing and compared the use of imputed LR genotyping verses HR genotyping for PIRCHE-II analysis and outcomes. Imputation success was highly dependent on the reference population used, as using historic Caucasian reference populations resulted in 10 % of pairs with unsuccessful imputation while multiethnic reference populations improved successful imputation with only 1 % unable to be imputed. Comparing PIRCHE-II analysis with HR and LR genotyping produced notably different results, with 20 % of patients discrepantly classified to immunologic risk groups. These data emphasize the importance of using multiethnic reference panels when performing imputation and indicate HR HLA genotyping has clinically meaningful benefit for PIRCHE-II analysis compared to imputed LR typing., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: G.P.M. has received research funding from CareDx, and research funding and travel support from Thermo-Fisher/One Lambda. C.C. has received research funding from PIRCHE AG. M.N. and B.D. are employees of PIRCHE AG., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Genetic diversity is critical to crop breeding and improvement, and dissection of the genomic variation underlying agronomic traits can both assist breeding and give insight into basic biological mechanisms. Although recent genome analyses in plants reveal many structural variants (SVs), most current studies of crop genetic variation are dominated by single-nucleotide polymorphisms (SNPs). The extent of the impact of SVs on global trait variation, as well as their utility in genome-wide selection, is not yet understood. In this study, we built an SV data set based on whole-genome resequencing of diverse sorghum lines (n = 363), validated the correlation of photoperiod sensitivity and variety type, and identified SV hotspots underlying the divergent evolution of cellulosic and sweet sorghum. In addition, we showed the complementary contribution of SVs for heritability of traits related to sorghum adaptation. Importantly, inclusion of SV polymorphisms in association studies revealed genotype-phenotype associations not observed with SNPs alone. Three-way genome-wide association studies (GWAS) based on whole-genome SNP, SV, and integrated SNP + SV data sets showed substantial associations between SVs and sorghum traits. The addition of SVs to GWAS substantially increased heritability estimates for some traits, indicating their important contribution to functional allelic variation at the genome level. Our discovery of the widespread impacts of SVs on heritable gene expression variation could render a plausible mechanism for their disproportionate impact on phenotypic variation. This study expands our knowledge of SVs and emphasizes the extensive impacts of SVs on sorghum., (© 2024 Zhang et al.; Published by Cold Spring Harbor Laboratory Press.)

Aims: Several modes of assay interference common to immunoassays affect solid-phase single-antigen bead-based immunoassays (SAB) used to detect antibodies against human leucocyte antigens (HLA). Best practice recommendations include methods to address assay interference, though the clinical impact and optimal approaches are undefined. We sought to evaluate assay interference in HLA SAB to identify an efficient approach for avoiding erroneous results., Methods: Retrospective analysis of 14 059 patient samples tested for anti-HLA antibodies was performed. This included 4685 samples tested prior to implementation of serum pretreatment with EDTA and 4982 samples tested with routine EDTA treatment using the same testing algorithm. An algorithm for efficiently identifying and processing samples with suspected interference was evaluated in a separate cohort of 4392 EDTA-treated samples., Results: EDTA serum pretreatment reduced assay interference, but did not eliminate all modes of interference. A protocol for identification and testing of samples with suspected interference facilitated efficient detection of interference while reducing the amount of additional testing required., Conclusions: Our data indicate that a single-method approach is insufficient to address all sources of interference in HLA SAB. A multimodal approach with a proactive screening is a more effective way to minimise risk of erroneous results., Competing Interests: Competing interests: GPM is a Scientific Advisory Board member for Transplant Diagnostics for Thermo-Fisher/One Lambda and has received research and travel support from Thermo-Fisher/One Lambda., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs)., Methods: We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively., Results: iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and a PDGF receptor-beta inhibitor impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive mediators., Conclusions: iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases., (© 2024. The Author(s).)

ABO blood group antigens are critical determinants of immunologic self and non-self and are ubiquitously expressed on all cellular tissues. Antibodies against non-self ABO antigens are naturally present and can mediate pathologic reactions against incompatible transfused blood cells and transplanted tissues. Laboratory testing for ABO antigens and isoagglutinins is essential for safe and effective transfusion and transplantation. Testing for ABO antigens has traditionally depended on serologic testing. However, there is increasing need for evaluation of genetic analysis of ABO antigens, to enable evaluation of ABO blood group in cases where serologic testing may be ambiguous or impossible to accurately perform. The clinical need for ABO genotyping is being addressed by the development of multiple molecular diagnostic approaches. Recent data have clearly demonstrated the potential utility of ABO genotyping in solid organ transplantation, yet widespread implementation has been slow. We propose that this lag is related to practical considerations in laboratory testing, including limited regulatory guidance on the performance and reporting of these assays and the absence of widely available external proficiency testing programs for quality assurance. Here we describe approaches to ABO genotyping, current initiatives in developing ABO genotyping proficiency testing programs, and laboratory quality assurance considerations for ABO genotyping., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: G.P.M. is a current Member of the College of American Pathologists Histocompatibility and Identity Testing Committee, Member of the Organ Procurement and Transplantation Network Histocompatibility Committee, and has received research and travel support from the National Institutes of Health, Thermo-Fisher/One Lambda, CareDx, and PIRCHE. C.M is currently a member and President Elect of the American Society for Histocompatibility and Immunogenetics (ASHI) and is also co-chair of the Luminex HLA advisory board., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Chilling tolerance in crops can increase resilience through longer growing seasons, drought escape, and nitrogen use efficiency. In sorghum (Sorghum bicolor [L.] Moench), breeding for chilling tolerance has been stymied by coinheritance of the largest-effect chilling tolerance locus, qSbCT04.62, with the major gene underlying undesirable grain proanthocyanidins, WD40 transcriptional regulator Tannin1. To test if this coinheritance is due to antagonistic pleiotropy of Tannin1, we developed and studied near-isogenic lines (NILs) carrying chilling tolerant haplotypes at qCT04.62. Whole-genome sequencing of the NILs revealed introgressions spanning part of the qCT04.62 confidence interval, including the Tannin1 gene and an ortholog of Arabidopsis cold regulator CBF/DREB1G. Segregation pattern of grain tannin in NILs confirmed the presence of wildtype Tannin1 and the reconstitution of a functional MYB-bHLH-WD40 regulatory complex. Low-temperature germination did not differ between NILs, suggesting that Tannin1 does not modulate this component of chilling tolerance. Similarly, NILs did not differ in seedling growth rate under either of two contrasting controlled environment chilling scenarios. Finally, while the chilling tolerant parent line had notably different photosynthetic responses from the susceptible parent line - including greater non-photochemical quenching before, during, and after chilling - the NIL responses match the susceptible parent. Thus, our findings suggest that tight linkage drag, not pleiotropy, underlies the precise colocalization of Tan1 with qCT04.62 and the qCT04.62 quantitative trait nucleotide lies outside the NIL introgressions. Breaking linkage at this locus should advance chilling tolerance breeding in sorghum and the identification of a novel chilling tolerance regulator., (© 2024. The Author(s).)

Breeding sorghum to withstand droughts is pivotal to secure crop production in regions vulnerable to water scarcity. Limited transpiration (LT) restricts water demand at high vapor pressure deficit, saving water for use in critical periods later in the growing season. Here we evaluated the hypothesis that LT would increase sorghum grain yield in the United States. We used a process-based crop model, APSIM, which simulates interactions of genotype, environment, and management (G × E × M). In this study, the G component includes the LT trait (G T ) and maturity group (G M ), the E W component entails water deficit patterns, and the M P component represents different planting dates. Simulations were conducted over 33 years (1986-2018) for representative locations across the US sorghum belt (Kansas, Texas, and Colorado) for three planting dates and maturity groups. The interaction of G T x E W indicated a higher impact of LT sorghum on grain for late drought (LD), mid-season drought (MD), and early drought (ED, 8%), than on well-watered (WW) environments (4%). Thus, significant impacts of LT can be achieved in western regions of the sorghum belt. The lack of interaction of G T × G M × M P suggested that an LT sorghum would increase yield by around 8% across maturity groups and planting dates. Otherwise, the interaction G M × M P revealed that specific combinations are better suited across geographical regions. Overall, the findings suggest that breeding for LT would increase sorghum yield in the drought-prone areas of the US without tradeoffs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer VJ declared a shared affiliation with the author GM to the handling editor at the time of review., (Copyright © 2024 Raymundo, Mclean, Sexton-Bowser, Lipka and Morris.)

Introduction: Accurate modelling of molecular changes in Alzheimer's disease (AD) dementia is crucial for understanding the mechanisms driving neuronal pathology and for developing treatments. Synaptic dysfunction has long been implicated as a mechanism underpinning memory dysfunction in AD and may result in part from changes in adenosine deaminase acting on RNA (ADAR) mediated RNA editing of the GluA2 subunit of AMPA receptors and changes in AMPA receptor function at the post synaptic cleft. However, few studies have investigated changes in proteins which influence RNA editing and notably, AD studies that focus on studying changes in protein expression, rather than changes in mRNA, often use traditional western blotting., Methods: Here, we demonstrate the value of automated capillary western blotting to investigate the protein expression of AMPA receptor subunits (GluA1-4), the ADAR RNA editing proteins (ADAR1-3), and proteins known to regulate RNA editing (PIN1, WWP2, FXR1P, and CREB1), in the J20 AD mouse model. We describe extensive optimisation and validation of the automated capillary western blotting method, demonstrating the use of total protein to normalise protein load, in addition to characterising the optimal protein/antibody concentrations to ensure accurate protein quantification. Following this, we assessed changes in proteins of interest in the hippocampus of 44-week-old J20 AD mice., Results: We observed an increase in the expression of ADAR1 p110 and GluA3 and a decrease in ADAR2 in the hippocampus of 44-week-old J20 mice. These changes signify a shift in the balance of proteins that play a critical role at the synapse. Regression analysis revealed unique J20-specific correlations between changes in AMPA receptor subunits, ADAR enzymes, and proteins that regulate ADAR stability in J20 mice, highlighting potential mechanisms mediating RNA-editing changes found in AD., Discussion: Our findings in J20 mice generally reflect changes seen in the human AD brain. This study underlines the importance of novel techniques, like automated capillary western blotting, to assess protein expression in AD. It also provides further evidence to support the hypothesis that a dysregulation in RNA editing-related proteins may play a role in the initiation and/or progression of AD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Milham, Morris, Konen, Rentsch, Avgan and Vissel.)

The Histocompatibility and Identity Testing Committee offers an overview of the College of American Pathologists' (CAP) Proficiency Testing (PT) program, commemorating its significant 75th anniversary in 2024. The CAP PT program has undergone significant growth and evolution over the years, ultimately achieving Centers for Medicare and Medicaid Services approval. In 1979, CAP's partnership with the American Association for Clinical Histocompatibility Testing marked a pivotal moment, leading to the creation of the first proficiency testing survey in 1980. This laid the foundation for various PT programs managed by the CAP Histocompatibility and Identity Testing Committee, including HLA antibody testing, HLA molecular typing, engraftment monitoring, parentage/relationship testing, HLA disease associations and drug risk, and HLA-B27 typing. Each program's distinctive considerations, grading methodologies, and future prospects are detailed here, highlighting the continual evolution of histocompatibility and identity testing PT to support emerging technologies and evolving laboratory practices in the field., Competing Interests: Author GcM was employed by the Laboratory Corporation of America Holdings. Author JS was employed by the Versiti Wisconsin Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sullivan, Gandhi, Gaitonde, Narasimhan, Gendzekhadze, Pandey, Roby, Maha, Kaur, Schiller, McDowell, Smith, Liu and Morris.)

Introduction: The impact of each immunosuppressive agent on de novo donor-specific antibodies in kidney transplant recipients varies among extant literature. Project aims: Patterns in immunosuppression and the effects on incidence of de novo donor-specific antibodies were evaluated. Design: Adult kidney transplant recipients from 2017 to 2019 without preformed antibodies were sampled. Allograft function, de novo donor-specific antibodies, tacrolimus concentrations, duration of goal-dose antiproliferatives, and steroid doses were recorded. Outcomes included incidence of de novo donor-specific antibodies, and their relation to tacrolimus concentrations, time at goal-dose antiproliferatives, and steroid doses. Results: Recipients (N = 153) were followed for 1 year; all were crossmatch negative and received rabbit antithymocyte globulin induction. Sixteen (10%) recipients developed de novo donor-specific antibodies in a median of 31 days [interquartile range, IQR: 12-67 days], most were Class II antibodies (87.5%). Incidence of de novo donor-specific antibodies did not differ based on induction dosing. Tacrolimus levels in the first month were lower for patients with de novo donor-specific antibodies (8.8 ng/mL vs 10.4 ng/mL, P < .01). There was no difference in time on goal antiproliferative doses, but higher steroid doses (0.4 vs 0.3 mg/kg/d; P = .02) were noted in patients with antibodies. Steroid dosing was likely impacted by baseline risk factors. Conclusion: A significant association was found between lower tacrolimus concentrations early post-transplant and incidence of de novo donor-specific antibodies. This highlighted the importance of clinician attention to subtle changes in tacrolimus and the impact it can have on antibody risk in the early post-transplant period., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

In the current genomic era, the search and deployment of new semi-dwarf alleles have continued to develop better plant types in all cereals. We characterized an agronomically optimal semi-dwarf mutation in Zea mays L. and a parallel polymorphism in Sorghum bicolor L. We cloned the maize brachytic1 (br1-Mu) allele by a modified PCR-based Sequence Amplified Insertion Flanking Fragment (SAIFF) approach. Histology and RNA-Seq elucidated the mechanism of semi-dwarfism. GWAS linked a sorghum plant height QTL with the Br1 homolog by resequencing a West African sorghum landraces panel. The semi-dwarf br1-Mu allele encodes an MYB transcription factor78 that positively regulates stalk cell elongation by interacting with the polar auxin pathway. Semi-dwarfism is due to differential splicing and low functional Br1 wild-type transcript expression. The sorghum ortholog, SbBr1, co-segregates with the major plant height QTL qHT7.1 and is alternatively spliced. The high frequency of the Sbbr1 allele in African landraces suggests that African smallholder farmers used the semi-dwarf allele to improve plant height in sorghum long before efforts to introduce Green Revolution-style varieties in the 1960s. Surprisingly, variants for differential splicing of Brachytic1 were found in both commercial maize and smallholder sorghum, suggesting parallel tuning of plant architecture across these systems., (© 2023 Corteva Agriscience. New Phytologist © 2023 New Phytologist Foundation.)

Competing Interests: None

The wild Tasmanian devil (Sarcophilus harrisii) population has suffered a devastating decline due to two clonal transmissible cancers. The first devil facial tumor 1 (DFT1) was observed in 1996, followed by a second genetically distinct transmissible tumor, the devil facial tumor 2 (DFT2), in 2014. DFT1/2 frequently metastasize, with lymph nodes being common metastatic sites. MHC-I downregulation by DFT1 cells is a primary means of evading allograft immunity aimed at polymorphic MHC-I proteins. DFT2 cells constitutively express MHC-I, and MHC-I is upregulated on DFT1/2 cells by interferon gamma, suggesting other immune evasion mechanisms may contribute to overcoming allograft and anti-tumor immunity. Human clinical trials have demonstrated PD1/PDL1 blockade effectively treats patients showing increased expression of PD1 in tumor draining lymph nodes, and PDL1 on peritumoral immune cells and tumor cells. The effects of DFT1/2 on systemic immunity remain largely uncharacterized. This study applied the open-access software QuPath to develop a semiautomated pipeline for whole slide analysis of stained tissue sections to quantify PD1/PDL1 expression in devil lymph nodes. The QuPath protocol provided strong correlations to manual counting. PD-1 expression was approximately 10-fold higher than PD-L1 expression in lymph nodes and was primarily expressed in germinal centers, whereas PD-L1 expression was more widely distributed throughout the lymph nodes. The density of PD1 positive cells was increased in lymph nodes containing DFT2 metastases, compared to DFT1. This suggests PD1/PDL1 exploitation may contribute to the poorly immunogenic nature of transmissible tumors in some devils and could be targeted in therapeutic or prophylactic treatments. Abbreviations: PD1: programmed cell death protein 1; PDL1: programmed death ligand 1; DFT1: devil facial tumor 1; DFT2: devil facial tumor 2; DFTD: devil facial tumor disease; MCC: Matthew's correlation coefficient; DAB: diaminobenzidine; ROI: region of interest.

Background: RNA editing at the Q/R site of GluA2 occurs with ~99% efficiency in the healthy brain, so that the majority of AMPARs contain GluA2(R) instead of the exonically encoded GluA2(Q). Reduced Q/R site editing infcreases AMPA receptor calcium permeability and leads to dendritic spine loss, neurodegeneration, seizures and learning impairments. Furthermore, GluA2 Q/R site editing is impaired in Alzheimer's disease (AD), raising the possibility that unedited GluA2(Q)-containing AMPARs contribute to synapse loss and neurodegeneration in AD. If true, then inhibiting expression of unedited GluA2(Q), while maintaining expression of GluA2(R), may be a novel strategy of preventing synapse loss and neurodegeneration in AD., Methods: We engineered mice with the 'edited' arginine codon (CGG) in place of the unedited glutamine codon (CAG) at position 607 of the Gria2 gene. We crossbred this line with the J20 mouse model of AD and conducted anatomical, electrophysiological and behavioural assays to determine the impact of eliminating unedited GluA2(Q) expression on AD-related phenotypes., Results: Eliminating unedited GluA2(Q) expression in AD mice prevented dendritic spine loss and hippocampal CA1 neurodegeneration as well as improved working and reference memory in the radial arm maze. These phenotypes were improved independently of Aβ pathology and ongoing seizure susceptibility. Surprisingly, our data also revealed increased spine density in non-AD mice with exonically encoded GluA2(R) as compared to their wild-type littermates, suggesting an unexpected and previously unknown role for unedited GluA2(Q) in regulating dendritic spines., Conclusion: The Q/R editing site of the AMPA receptor subunit GluA2 may act as an epigenetic switch that regulates dendritic spines, neurodegeneration and memory deficits in AD., (© 2023. Editorial Group and BioMed Central Ltd., part of Springer Nature.)

In temperate climates, earlier planting of tropical-origin crops can provide longer growing seasons, reduce water loss, suppress weeds, and escape post-flowering drought stress. However, chilling sensitivity of sorghum, a tropical-origin cereal crop, limits early planting, and over 50 years of conventional breeding has been stymied by coinheritance of chilling tolerance (CT) loci with undesirable tannin and dwarfing alleles. In this study, phenomics and genomics-enabled approaches were used for prebreeding of sorghum early-season CT. Uncrewed aircraft systems (UAS) high-throughput phenotyping platform tested for improving scalability showed moderate correlation between manual and UAS phenotyping. UAS normalized difference vegetation index values from the chilling nested association mapping population detected CT quantitative trait locus (QTL) that colocalized with manual phenotyping CT QTL. Two of the 4 first-generation Kompetitive Allele Specific PCR (KASP) molecular markers, generated using the peak QTL single nucleotide polymorphisms (SNPs), failed to function in an independent breeding program as the CT allele was common in diverse breeding lines. Population genomic fixation index analysis identified SNP CT alleles that were globally rare but common to the CT donors. Second-generation markers, generated using population genomics, were successful in tracking the donor CT allele in diverse breeding lines from 2 independent sorghum breeding programs. Marker-assisted breeding, effective in introgressing CT allele from Chinese sorghums into chilling-sensitive US elite sorghums, improved early-planted seedling performance ratings in lines with CT alleles by up to 13-24% compared to the negative control under natural chilling stress. These findings directly demonstrate the effectiveness of high-throughput phenotyping and population genomics in molecular breeding of complex adaptive traits., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Cerebral blood flow (CBF) is important for the maintenance of brain function and its dysregulation has been implicated in Alzheimer's disease (AD). Microglia associations with capillaries suggest they may play a role in the regulation of CBF or the blood-brain-barrier (BBB). We explored the relationship between microglia and pericytes, a vessel-resident cell type that has a major role in the control of CBF and maintenance of the BBB, discovering a spatially distinct subset of microglia that closely associate with pericytes. We termed these pericyte-associated microglia (PEM). PEM are present throughout the brain and spinal cord in NG2DsRed × CX 3 CR1 +/GFP mice, and in the human frontal cortex. Using in vivo two-photon microscopy, we found microglia residing adjacent to pericytes at all levels of the capillary tree and found they can maintain their position for at least 28 days. PEM can associate with pericytes lacking astroglial endfeet coverage and capillary vessel width is increased beneath pericytes with or without an associated PEM, but capillary width decreases if a pericyte loses a PEM. Deletion of the microglia fractalkine receptor (CX 3 CR1) did not disrupt the association between pericytes and PEM. Finally, we found the proportion of microglia that are PEM declines in the superior frontal gyrus in AD. In summary, we identify microglia that specifically associate with pericytes and find these are reduced in number in AD, which may be a novel mechanism contributing to vascular dysfunction in neurodegenerative diseases., (© 2023 The Authors. GLIA published by Wiley Periodicals LLC.)

Autoimmunity and cancer represent two different aspects of immune dysfunction. Autoimmunity is characterized by breakdowns in immune self-tolerance, while impaired immune surveillance can allow for tumorigenesis. The class I major histocompatibility complex (MHC-I), which displays derivatives of the cellular peptidome for immune surveillance by CD8 + T cells, serves as a common genetic link between these conditions. As melanoma-specific CD8 + T cells have been shown to target melanocyte-specific peptide antigens more often than melanoma-specific antigens, we investigated whether vitiligo- and psoriasis-predisposing MHC-I alleles conferred a melanoma-protective effect. In individuals with cutaneous melanoma from both The Cancer Genome Atlas (n = 451) and an independent validation set (n = 586), MHC-I autoimmune-allele carrier status was significantly associated with a later age of melanoma diagnosis. Furthermore, MHC-I autoimmune-allele carriers were significantly associated with decreased risk of developing melanoma in the Million Veteran Program (OR = 0.962, p = 0.024). Existing melanoma polygenic risk scores (PRSs) did not predict autoimmune-allele carrier status, suggesting these alleles provide orthogonal risk-relevant information. Mechanisms of autoimmune protection were neither associated with improved melanoma-driver mutation association nor improved gene-level conserved antigen presentation relative to common alleles. However, autoimmune alleles showed higher affinity relative to common alleles for particular windows of melanocyte-conserved antigens and loss of heterozygosity of autoimmune alleles caused the greatest reduction in presentation for several conserved antigens across individuals with loss of HLA alleles. Overall, this study presents evidence that MHC-I autoimmune-risk alleles modulate melanoma risk unaccounted for by current PRSs., Competing Interests: Declaration of interests G.P.M. receives research support from Thermo Fisher, CareDx, and PIRCHE. M.Z. is a board member of Invectys Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Donor-derived cell-free DNA (dd-cfDNA) testing is an emerging screening modality for noninvasive detection of acute rejection (AR). This study compared the testing accuracy for AR of two commercially available dd-cfDNA and gene-expression profiling (GEP) testing in heart transplant (HTx) recipients., Methods: This is a retrospective, observational study of HTx only patients who underwent standard and expanded single nucleotide polymorphism (SNP) dd-cfDNA between October 2020 to January 2022. Comparison with GEP was also performed. Assays were compared for correlation, accurate classification, and prediction for AR., Results: A total of 428 samples from 112 unique HTx patients were used for the study. A positive standard SNP correlated with the expanded SNP assay (p < .001). Both standard and expanded SNP tests showed low sensitivity (39%, p = 1.0) but high specificity (82% and 84%, p = 1.0) for AR. GEP did not improve sensitivity and showed worse specificity (p < .001) compared to standard dd-cfDNA., Conclusion: We found no significant difference between standard and expanded SNP assays in detecting AR. We show improved specificity without change in sensitivity using dd-cfDNA in place of GEP testing. Prospective controlled studies to address how to best implement dd-cfDNA testing into clinical practice are needed., (© 2023 The Authors. Clinical Transplantation published by John Wiley & Sons Ltd.)

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can cure patients with high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). However, many patients relapse or develop debilitating graft-versus-host disease. Transplant restores T-cell reactivity against tumor cells, implicating patient human leukocyte antigen (HLA)-dependent antigen presentation via the major histocompatibility complex as a determinant of response. We sought to identify characteristics of the HLA genotype that influence response in allo-HSCT patients., Methods: We collected HLA genotype and panel-based somatic mutation profiles for 55 patients with AML and MDS and available data treated at the University of California San Diego Moores Cancer Center between May 2012 and January 2019. We evaluated characteristics of the HLA genotype relative to relapse-free time and overall survival (OS) post-allo-HSCT using univariable and multivariable regression., Results: In multivariable regression, the presence of an autoimmune allele was significantly associated with relapse-free time (hazard ratio [HR], 0.25; p = 0.01) and OS (HR, 0.16; p < 0.005). The better potential of the donor HLA type to present peptides harboring driver mutations trended toward better relapse-free survival (HR, 0.45; p = 0.07) and significantly correlated with longer OS (HR, 0.33; p = 0.01) though only a minority of cases had an HLA mismatch., Conclusion: In this single institution retrospective study of patients receiving allo-HSCT for relapsed AML/MDS, characteristics of an individual's HLA genotype (presence of an autoimmune allele and potential of the donor HLA to better present peptides representing driver mutations) were significantly associated with better outcomes. These findings suggest that HLA type may guide the optimal application of allo-HSCT and merit evaluation in larger cohorts. ClinicalTrials.gov Identifier: NCT02478931 ., Competing Interests: Conflict of Interest: Dr. Goodman is a consultant for Seattle Genetics. Garret Frampton is an employee of Foundation Medicine and a shareholder of Roche. Dr. Kurzrock has an equity interest in CureMatch Inc. and IDbyDNA; serves on the Board of CureMatch and CureMetrix, and is a co-founder of CureMatch.

With the continued promise of immunotherapy for treating cancer, understanding how host genetics contributes to the tumor immune microenvironment (TIME) is essential to tailoring cancer screening and treatment strategies. Here, we study 1084 eQTLs affecting the TIME found through analysis of The Cancer Genome Atlas and literature curation. These TIME eQTLs are enriched in areas of active transcription, and associate with gene expression in specific immune cell subsets, such as macrophages and dendritic cells. Polygenic score models built with TIME eQTLs reproducibly stratify cancer risk, survival and immune checkpoint blockade (ICB) response across independent cohorts. To assess whether an eQTL-informed approach could reveal potential cancer immunotherapy targets, we inhibit CTSS, a gene implicated by cancer risk and ICB response-associated polygenic models; CTSS inhibition results in slowed tumor growth and extended survival in vivo. These results validate the potential of integrating germline variation and TIME characteristics for uncovering potential targets for immunotherapy., (© 2023. The Author(s).)

Background: Discovery that ~16% of T cells naturally co-express two T-cell receptor (TCR) clonotypes prompts examining the role of dual TCR cells in immune functions., Methods: Using TCRα-reporter transgenic mice, enabling unambiguous identification of single-TCR and dual-TCR cells, we tested the role of dual TCR cells in antitumor immune responses against immune-responsive syngeneic 6727 sarcoma and immune-resistant B16F10 melanoma., Results: Dual TCR cells were specifically increased among tumor-infiltrating lymphocytes (TILs) in both models, indicating selective advantage in antitumor responses. Phenotype and single-cell gene expression analyses identified dual TCR are predominant during the effective antitumor response, demonstrating selectively increased activation in the TIL compartment and skewing toward an effector memory phenotype. Absence of dual TCR cells impaired immune response to B16F10 but not 6727, suggesting that dual TCR cells may be more influential in responses against poorly immunogenic tumors. Dual TCR cells demonstrated an advantage in recognition of B16F10-derived neoantigens in vitro, providing a mechanistic basis for their antitumor reactivity., Conclusions: These results discover an unrecognized role for dual TCR cells in protective immune function and identify these cells and their TCRs as a potential resource for antitumor immunotherapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Crop biofortification is a successful strategy to ameliorate Vitamin A deficiency. Sorghum is a good candidate for vitamin A biofortification, as it is a staple food in regions with high prevalence of vitamin A deficiency. β-carotene-the main provitamin A carotenoid-is below the target concentration in sorghum grain, therefore biofortification breeding is required. Previous studies found evidence that sorghum carotenoid variation is oligogenic, suggesting that marker-assisted selection can be an appropriate biofortification method. However, we hypothesize that sorghum carotenoids have both oligogenic and polygenic components of variation. Genomics-assisted breeding could accelerate breeding efforts, but there exists knowledge gaps in the genetics underlying carotenoid variation, as well as appropriate germplasm to serve as donors., Results: In this study, we characterized carotenoids in 446 accessions from the sorghum association panel and carotenoid panel using high-performance liquid chromatography, finding high carotenoid accessions not previously identified. Genome-wide association studies conducted with 345 accessions, confirmed that zeaxanthin epoxidase is a major gene underlying variation for not only zeaxanthin, but also lutein and β-carotene. High carotenoid lines were found to have limited genetic diversity, and originated predominantly from only one country. Potential novel genetic diversity for carotenoid content was identified through genomic predictions in 2,495 accessions of unexplored germplasm. Oligogenic variation of carotenoids was confirmed, as well as evidence for polygenic variation, suggesting both marker-assisted selection and genomic selection can facilitate breeding efforts., Conclusions: Sorghum vitamin A biofortification could be beneficial for millions of people who rely on it as a dietary staple. Carotenoid content in sorghum is low, but high heritability suggests that increasing concentrations through breeding is possible. Low genetic diversity among high carotenoid lines might be the main limitation for breeding efforts, therefore further germplasm characterization is needed to assess the feasibility of biofortification breeding. Based on germplasm here evaluated, most countries' germplasm lacks high carotenoid alleles, thus pre-breeding will be needed. A SNP marker within the zeaxanthin epoxidase gene was identified as a good candidate for use in marker-assisted selection. Due to the oligogenic and polygenic variation of sorghum grain carotenoids, both marker-assisted selection and genomic selection can be employed to accelerate breeding efforts., (© 2023. The Author(s).)

Stroke therapy has largely focused on preventing damage and encouraging repair outside the ischemic core, as the core is considered irreparable. Recently, several studies have suggested endogenous responses within the core are important for limiting the spread of damage and enhancing recovery, but the role of blood flow and capillary pericytes in this process is unknown. Using the Rose Bengal photothrombotic model of stroke, we illustrate blood vessels are present in the ischemic core and peri-lesional regions 2 weeks post stroke in male mice. A FITC-albumin gel cast of the vasculature revealed perfusion of these vessels, suggesting cerebral blood flow (CBF) may be partially present, without vascular leakage. The length of these vessels is significantly reduced compared to uninjured regions, but the average width is greater, suggesting they are either larger vessels that survived the initial injury, smaller vessels that have expanded in size (i.e., arteriogenesis), or that neovascularization begins with larger vessels. Concurrently, we observed an increase in platelet-derived growth factor receptor beta (PDGFRβ, a marker of pericytes) expression within the ischemic core in two distinct patterns, one which resembles pericyte-derived fibrotic scarring at the edge of the core, and one which is vessel associated and may represent blood vessel recovery. We find little evidence for dividing cells on these intralesional blood vessels 2 weeks post stroke. Our study provides evidence flow is present in PDGFRβ-positive vessels in the ischemic core 2 weeks post stroke. We hypothesize intralesional CBF is important for limiting injury and for encouraging endogenous repair following cerebral ischemia., (© 2022 The Authors. Journal of Neuroscience Research published by Wiley Periodicals LLC.)

A fundamental goal in plant biology is to identify and understand the variation underlying plants' adaptation to their environment. Climate change has given new urgency to this goal, as society aims to accelerate adaptation of ecologically important plant species, endangered plant species, and crops to hotter, less predictable climates. In the pre-genomic era, identifying adaptive alleles was painstaking work, leveraging genetics, molecular biology, physiology, and ecology. Now, the rise of genomics and new computational approaches may facilitate this research. Genotype-environment associations (GEAs) use statistical associations between allele frequency and environment of origin to test the hypothesis that allelic variation at a given gene is adapted to local environments. Researchers may scan the genome for GEAs to generate hypotheses on adaptive genetic variants (environmental genome-wide association studies). Despite the rapid adoption of these methods, many important questions remain about the interpretation of GEA findings, which arise from fundamental unanswered questions on the genetic architecture of adaptation and limitations inherent to association-based analyses. We outline strategies to ground GEAs in the underlying hypotheses of genetic architecture and better test GEA-generated hypotheses using genetics and ecophysiology. We provide recommendations for new users who seek to learn about the molecular basis of adaptation. When combined with a rigorous hypothesis testing framework, GEAs may facilitate our understanding of the molecular basis of climate adaptation for plant improvement., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To compare the effects of particle abrasion medium and pressure on shear bond strength and biaxial flexural strength of three generations of zirconia (Lava Frame, Lava Plus, and Lava Esthetic) with the goal of optimizing the bond to zirconia., Methods: 280 discs (14 mm diameter; 1 mm thickness) of each zirconia were milled and sintered. Specimens of each material were randomly distributed into 14 groups (n=20); half were tested for shear bond strength and half were tested for biaxial flexural strength. The specimens were particle abraded on one surface by 2 different media (50 μm alumina particles or 50 μm glass beads) for 10 seconds at three different pressures (15, 30, and 45 psi or 0.1, 0.2, 0.3 MPa). Untreated specimens served as positive control. A tube (1.50 mm diameter) filled with dual cured resin cement (Panavia SA) was placed onto the surface and light cured. Specimens were stored in water (37°C for 24 hours) and shear bond strength was measured in a universal testing machine (Instron). Biaxial flexural strength of each specimen was measured according to ISO 6872. Shear bond strength and biaxial flexural strength were compared individually with a 2-way analysis of variance (ANOVA) for factors surface treatment and zirconia composition., Results: Significant differences were seen between surface treatments (p<0.01), zirconia composition (p<0.01) and their interaction (p<0.01) for both bond strength and flexural strength. With alumina particle abrasion, higher pressure produced higher bonds for Lava Frame and Lava Plus zirconia while the bond of Lava Esthetic declined with increased pressure. Higher pressure (>0.2 MPa or 30 psi) with alumina decreased biaxial flexural strength with Lava Esthetic zirconia., Conclusions: Particle abrasion with alumina produced a significantly better combination of bond strength while maintaining biaxial strength of three zirconia materials than particle abrasion with glass beads. The bond strength also depended upon the pressure of particle abrasion and the generation of zirconia used., (© Operative Dentistry, 2023.)

Purpose: The contribution of common genetic variants to pre-cancer progression is understudied due to long follow-up time, rarity of poor outcomes and lack of available germline DNA collection. Alternatively, DNA from diagnostic archival tissue is available, but its somatic nature, limited quantity and suboptimal quality would require an accurate cost-effective genome-wide germline genotyping methodology., Experimental Design: Blood and tissue DNA from 10 individuals were used to benchmark the accuracy of Single Nucleotide Polymorphisms (SNP) genotypes, Polygenic Risk Scores (PRS) or HLA haplotypes using low-coverage whole-genome sequencing (lc-WGS) and genotype imputation. Tissue-derived PRS were further evaluated for 36 breast cancer patients (11.7 years median follow-up time) diagnosed with DCIS and used to model the risk of Breast Cancer Subsequent Events (BCSE)., Results: Tissue-derived germline DNA profiling resulted in accurate genotypes at common SNPs (blood correlation r 2  > 0.94) and across 22 disease-related polygenic risk scores (PRS, mean correlation r = 0.93). Imputed Class I and II HLA haplotypes were 96.7% and 82.5% concordant with clinical-grade blood HLA haplotypes, respectively. In DCIS patients, tissue-derived PRS was significantly associated with BCSE (HR = 2, 95% CI 1.2-3.8). The top and bottom decile patients had an estimated 28% and 5% chance of BCSE at 10 years, respectively., Conclusions: Archival tissue DNA germline profiling using lc-WGS and imputation, represents a cost and resource-effective alternative in the retrospective design of long-term disease genetic studies. Initial results in breast cancer suggest that common risk variants contribute to pre-cancer progression., (© 2022. The Author(s).)