POT1 recruits and regulates CST-Polα/primase at human telomeres.

Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.

Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses.

Several ebolaviruses cause outbreaks of severe disease. Vaccines and monoclonal antibody cocktails are available to treat Ebola virus (EBOV) infections, but not Sudan virus (SUDV) or other ebolaviruses. Current cocktails contain antibodies that cross-react with the secreted soluble glycoprotein (sGP) that absorbs virus-neutralizing antibodies. By sorting memory B cells from EBOV infection survivors, we isolated two broadly reactive anti-GP monoclonal antibodies, 1C3 and 1C11, that potently neutralize, protect rodents from disease, and lack sGP cross-reactivity. Both antibodies recognize quaternary epitopes in trimeric ebolavirus GP. 1C11 bridges adjacent protomers via the fusion loop. 1C3 has a tripartite epitope in the center of the trimer apex. One 1C3 antigen-binding fragment anchors simultaneously to the three receptor-binding sites in the GP trimer, and separate 1C3 paratope regions interact differently with identical residues on the three protomers. A cocktail of both antibodies completely protected nonhuman primates from EBOV and SUDV infections, indicating their potential clinical value.

Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection.

Ebola virus (EBOV) remains a public health threat. We performed a longitudinal study of B cell responses to EBOV in four survivors of the 2014 West African outbreak. Infection induced lasting EBOV-specific immunoglobulin G (IgG) antibodies, but their subclass composition changed over time, with IgG1 persisting, IgG3 rapidly declining, and IgG4 appearing late. Striking changes occurred in the immunoglobulin repertoire, with massive recruitment of naive B cells that subsequently underwent hypermutation. We characterized a large panel of EBOV glycoprotein-specific monoclonal antibodies (mAbs). Only a small subset of mAbs that bound glycoprotein by ELISA recognized cell-surface glycoprotein. However, this subset contained all neutralizing mAbs. Several mAbs protected against EBOV disease in animals, including one mAb that targeted an epitope under evolutionary selection during the 2014 outbreak. Convergent antibody evolution was seen across multiple donors, particularly among VH3-13 neutralizing antibodies specific for the GP1 core. Our study provides a benchmark for assessing EBOV vaccine-induced immunity.

Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres.

Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association. Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT with the telomeric protein TPP1. Our results reveal that telomerase recruitment to telomeres is driven by dynamic interactions between the rapidly diffusing telomerase and the chromosome end.

Site-specific reactivity of stepped Pt surfaces driven by stress release.

Heterogeneous catalysts are widely used to promote chemical reactions. Although it is known that chemical reactions usually happen on catalyst surfaces, only specific surface sites have high catalytic activity. Thus, identifying active sites and maximizing their presence lies at the heart of catalysis research1-4, in which the classic model is to categorize active sites in terms of distinct surface motifs, such as terraces and steps1,5-10. However, such a simple categorization often leads to orders of magnitude errors in catalyst activity predictions and qualitative uncertainties of active sites7,8,11,12, thus limiting opportunities for catalyst design. Here, using stepped Pt(111) surfaces and the electrochemical oxygen reduction reaction (ORR) as examples, we demonstrate that the root cause of larger errors and uncertainties is a simplified categorization that overlooks atomic site-specific reactivity driven by surface stress release. Specifically, surface stress release at steps introduces inhomogeneous strain fields, with up to 5.5% compression, leading to distinct electronic structures and reactivity for terrace atoms with identical local coordination, and resulting in atomic site-specific enhancement of ORR activity. For the terrace atoms flanking both sides of the step edge, the enhancement is up to 50 times higher than that of the atoms in the middle of the terrace, which permits control of ORR reactivity by either varying terrace widths or controlling external stress. Thus, the discovery of the above synergy provides a new perspective for both fundamental understanding of catalytically active atomic sites and design principles of heterogeneous catalysts.

Reconstitution of a telomeric replicon organized by CST.

Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins1. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells2. This replication requires telomerase3 extension of the single-stranded DNA (ssDNA) of the telomeric G-strand ((TTAGGG)n); the synthesis of the complementary C-strand ((CCCTAA)n) is much less well characterized. The CST (CTC1-STN1-TEN1) protein complex, a DNA polymerase α-primase accessory factor4,5, is known to be required for telomere replication in vivo6-9, and the molecular analysis presented here reveals key features of its mechanism. We find that human CST uses its ssDNA-binding activity to specify the origins for telomeric C-strand synthesis by bound Polα-primase. CST-organized DNA polymerization can copy a telomeric DNA template that folds into G-quadruplex structures, but the challenges presented by this template probably contribute to telomere replication problems observed in vivo. Combining telomerase, a short telomeric ssDNA primer and CST-Polα-primase gives complete telomeric DNA replication, resulting in the same sort of ssDNA 3' overhang found naturally on human telomeres. We conclude that the CST complex not only terminates telomerase extension10,11 and recruits Polα-primase to telomeric ssDNA4,12,13 but also orchestrates C-strand synthesis. Because replication of the telomere has features distinct from replication of the rest of the genome, targeting telomere-replication components including CST holds promise for cancer therapeutics.

Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB.

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity1-4. The ms2i6A modification is installed onto isopentenyladenosine (i6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6-9 in sulfur transfer to C2 of i6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C2 hydrogen of the substrate but only after C2 has undergone rehybridization from sp2 to sp3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.

Smallpox lesion characterization in placebo-treated and tecovirimat-treated macaques using traditional and novel methods.

Smallpox was the most rampant infectious disease killer of the 20th century, yet much remains unknown about the pathogenesis of the variola virus. Using archived tissue from a study conducted at the Centers for Disease Control and Prevention we characterized pathology in 18 cynomolgus macaques intravenously infected with the Harper strain of variola virus. Six macaques were placebo-treated controls, six were tecovirimat-treated beginning at 2 days post-infection, and six were tecovirimat-treated beginning at 4 days post-infection. All macaques were treated daily until day 17. Archived tissues were interrogated using immunohistochemistry, in situ hybridization, immunofluorescence, and electron microscopy. Gross lesions in three placebo-treated animals that succumbed to infection primarily consisted of cutaneous vesicles, pustules, or crusts with lymphadenopathy. The only gross lesions noted at the conclusion of the study in the three surviving placebo-treated and the Day 4 treated animals consisted of resolving cutaneous pox lesions. No gross lesions attributable to poxviral infection were present in the Day 2 treated macaques. Histologic lesions in three placebo-treated macaques that succumbed to infection consisted of proliferative and necrotizing dermatitis with intracytoplasmic inclusion bodies and lymphoid depletion. The only notable histologic lesion in the Day 4 treated macaques was resolving dermatitis; no notable lesions were seen in the Day 2 treated macaques. Variola virus was detected in all three placebo-treated animals that succumbed to infection prior to the study's conclusion by all utilized methods (IHC, ISH, IFA, EM). None of the three placebo-treated animals that survived to the end of the study nor the animals in the two tecovirimat treatment groups showed evidence of variola virus by these methods. Our findings further characterize variola lesions in the macaque model and describe new molecular methods for variola detection.

A hypothalamic novelty signal modulates hippocampal memory.

The ability to recognize information that is incongruous with previous experience is critical for survival. Novelty signals have therefore evolved in the mammalian brain to enhance attention, perception and memory1,2. Although the importance of regions such as the ventral tegmental area3,4 and locus coeruleus5 in broadly signalling novelty is well-established, these diffuse monoaminergic transmitters have yet to be shown to convey specific information on the type of stimuli that drive them. Whether distinct types of novelty, such as contextual and social novelty, are differently processed and routed in the brain is unknown. Here we identify the supramammillary nucleus (SuM) as a novelty hub in the hypothalamus6. The SuM region is unique in that it not only responds broadly to novel stimuli, but also segregates and selectively routes different types of information to discrete cortical targets-the dentate gyrus and CA2 fields of the hippocampus-for the modulation of mnemonic processing. Using a new transgenic mouse line, SuM-Cre, we found that SuM neurons that project to the dentate gyrus are activated by contextual novelty, whereas the SuM-CA2 circuit is preferentially activated by novel social encounters. Circuit-based manipulation showed that divergent novelty channelling in these projections modifies hippocampal contextual or social memory. This content-specific routing of novelty signals represents a previously unknown mechanism that enables the hypothalamus to flexibly modulate select components of cognition.

Lignin with controlled structural properties by N-heterocycle-based deep eutectic solvent extraction.

The complex and heterogeneous nature of the lignin macromolecule has presented a lasting barrier to its utilization. To achieve high lignin yield, the technical lignin extraction process usually severely modifies and condenses the native structure of lignin, which is a critical drawback for its utilization in conversion processes. In addition, there is no method capable of separating lignin from plant biomass with controlled structural properties. Here, we developed an N-heterocycle-based deep eutectic solvent formed between lactic acid and pyrazole (La-Py DES) with a binary hydrogen bonding functionality resulting in a high affinity toward lignin. Up to 93.7% of lignin was extracted from wheat straw biomass at varying conditions from 90 °C to 145 °C. Through careful selection of treatment conditions as well as lactic acid to pyrazole ratios, lignin with controlled levels of ether linkage content, hydroxyl group content, and average molecular weight can be generated. Under mild extraction conditions (90 °C to 120 °C), light-colored native-like lignin can be produced with up to 80% yield, whereas ether linkage-free lignin with low polydispersity can be obtained at 145 °C. Overall, this study offers a new strategy for native lignin extraction and generating lignin with controlled structural properties.

FOXO inhibition rescues α-defensin expression in human intestinal organoids.

To mediate critical host-microbe interactions in the human small intestine, Paneth cells constitutively produce abundant levels of α-defensins and other antimicrobials. We report that the expression profile of these antimicrobials is dramatically askew in human small intestinal organoids (enteroids) as compared to that in paired tissue from which they are derived, with a reduction of α-defensins to nearly undetectable levels. Murine enteroids, however, recapitulate the expression profile of Paneth cell α-defensins seen in tissue. WNT/TCF signaling has been found to be instrumental in the regulation of α-defensins, yet in human enteroids exogenous stimulation of WNT signaling appears insufficient to rescue α-defensin expression. By stark contrast, forkhead box O (FOXO) inhibitor AS1842856 induced the expression of α-defensin mRNA in enteroids by >100,000-fold, restoring DEFA5 and DEFA6 to levels comparable to those found in primary human tissue. These results newly identify FOXO signaling as a pathway of biological and potentially therapeutic relevance for the regulation of human Paneth cell α-defensins in health and disease.

Cholangiocytes express an isoform of soluble adenylyl cyclase that is N-linked glycosylated and secreted in extracellular vesicles.

Soluble adenylyl cyclase (sAC)-derived cAMP regulates various cellular processes; however, the regulatory landscape mediating sAC protein levels remains underexplored. We consistently observed a 85 kD (sAC85 ) or 75 kD (sAC75 ) sAC protein band under glucose-sufficient or glucose-deprived states, respectively, in H69 cholangiocytes by immunoblotting. Deglycosylation by PNGase-F demonstrated that both sAC75 and sAC85 are N-linked glycosylated proteins with the same polypeptide backbone. Deglycosylation with Endo-H further revealed that sAC75 and sAC85 carry distinct sugar chains. We observed release of N-linked glycosylated sAC (sACEV ) in extracellular vesicles under conditions that support intracellular sAC85 (glucose-sufficient) as opposed to sAC75 (glucose-deprived) conditions. Consistently, disrupting the vesicular machinery affects the maturation of intracellular sAC and inhibits the release of sACEV into extracellular vesicles. The intracellular turnover of sAC85 is extremely short (t1/2 ~30 min) and release of sACEV in the medium was detected within 3 h. Our observations support the maturation and trafficking in cholangiocytes of an N-linked glycosylated sAC isoform that is rapidly released into extracellular vesicles.

Reconstitution of the alternative pathway of the complement system enables rapid delineation of the mechanism of action of novel inhibitors.

The complement system plays a critical role in the innate immune response, acting as a first line of defense against invading pathogens. However, dysregulation of the complement system is implicated in the pathogenesis of numerous diseases, ranging from Alzheimer's to age-related macular degeneration and rare blood disorders. As such, complement inhibitors have enormous potential to alleviate disease burden. While a few complement inhibitors are in clinical use, there is still a significant unmet medical need for the discovery and development of novel inhibitors to treat patients suffering from disorders of the complement system. A key hurdle in the development of complement inhibitors has been the determination of their mechanism of action. Progression along the complement cascade involves the formation of numerous multimeric protein complexes, creating the potential for inhibitors to act at multiple nodes in the pathway. This is especially true for molecules that target the central component C3 and its fragment C3b, which serve a dual role as a substrate for the C3 convertases and as a scaffolding protein in both the C3 and C5 convertases. Here, we report a step-by-step in vitro reconstitution of the complement alternative pathway using bio-layer interferometry. By physically uncoupling each step in the pathway, we were able to determine the kinetic signature of inhibitors that act at single steps in the pathway and delineate the full mechanism of action of known and novel C3 inhibitors. The method could have utility in drug discovery and further elucidating the biochemistry of the complement system.

Art and Science of the Cellular Mesoscale.

Experimental information from microscopy, structural biology, and bioinformatics may be integrated to build structural models of entire cells with molecular detail. This integrative modeling is challenging in several ways: the intrinsic complexity of biology results in models with many closely packed and heterogeneous components; the wealth of available experimental data is scattered among multiple resources and must be gathered, reconciled, and curated; and computational infrastructure is only now gaining the capability of modeling and visualizing systems of this complexity. We present recent efforts to address these challenges, both with artistic approaches to depicting the cellular mesoscale, and development and application of methods to build quantitative models.

Optimizing Spectronaut Search Parameters to Improve Data Quality with Minimal Proteome Coverage Reductions in DIA Analyses of Heterogeneous Samples.

Data-independent acquisition has seen breakthroughs that enable comprehensive proteome profiling using short gradients. As the proteome coverage continues to increase, the quality of the data generated becomes much more relevant. Using Spectronaut, we show that the default search parameters can be easily optimized to minimize the occurrence of false positives across different samples. Using an immunological infection model system to demonstrate the impact of adjusting search settings, we analyzed Mus musculus macrophages and compared their proteome to macrophages spiked withCandida albicans. This experimental system enabled the identification of "false positives" as Candida albicans peptides and proteins should not be present in the Mus musculus-only samples. We show that adjusting the search parameters reduced "false positive" identifications by 89% at the peptide and protein level, thereby considerably increasing the quality of the data. We also show that these optimized parameters incurred a moderate cost, only reducing the overall number of "true positive" identifications across each biological replicate by <6.7% at both the peptide and protein level. We believe the value of our updated search parameters extends beyond a two-organism analysis and would be of great value to any DIA experiment analyzing heterogeneous populations of cell types or tissues.

Piperine enhances contractile force in slow- and fast-twitch muscle.

Piperine has been shown to bind to myosin and shift the distribution of conformational states of myosin molecules from the super-relaxed state to the disordered relaxed state. However, little is known about the implications for muscle force production and potential underlying mechanisms. Muscle contractility experiments were performed using isolated muscles and single fibres from rats and mice. The dose-response effect of piperine on muscle force was assessed at several stimulation frequencies. The potentiation of muscle force was also tested in muscles fatigued by eccentric contractions. Potential mechanisms of force potentiation were assessed by measuring Ca2+ levels during stimulation in enzymatically dissociated muscle fibres, while myofibrillar Ca2+ sensitivity was assessed in chemically skinned muscle fibres. Piperine caused a dose-dependent increase in low-frequency force with no effect on high-frequency force in both slow- and fast-twitch muscle, with similar relative increases in twitch force, rate of force development and relaxation rate. The potentiating effect of piperine on low-frequency force was reversible, and piperine partially recovered low-frequency force in fatigued muscle. Piperine had no effect on myoplasmic free [Ca2+] levels in mouse muscle fibres, whereas piperine substantially augmented the force response to submaximal levels of [Ca2+] in rat MyHCII fibres and MyHCI fibres along with a minor increase in maximum Ca2+-activated force. Piperine enhances low-frequency force production in both fast- and slow-twitch muscle. The effects are reversible and can counteract muscle fatigue. The primary underlying mechanism appears to be an increase in Ca2+ sensitivity. KEY POINTS: Piperine is a plant alkaloid derived from black pepper. It is known to bind to skeletal muscle myosin and enhance resting ATP turnover but its effects on contractility are not well known. We showed for the first time a piperine-induced force potentiation that was pronounced during low-frequency electrical stimulation of isolated muscles. The effect of piperine was observed in both slow and fast muscle types, was reversible, and could counteract the force decrements observed after fatiguing muscle contractions. Piperine treatment caused an increase in myofibrillar Ca2+ sensitivity in chemically skinned muscle fibres, while we observed no effect on intracellular Ca2+ concentrations during electrical stimulation in enzymatically dissociated muscle fibres.

Anesthetic Care of the Pregnant Patient With Cardiovascular Disease: A Scientific Statement From the American Heart Association.

The pregnancy-related mortality rate in the United States is excessively high. The American Heart Association is dedicated to fighting heart disease and recognizes that cardiovascular disease, preexisting or acquired during pregnancy, is the leading cause of maternal mortality in the United States. Comprehensive scientific statements from cardiology and obstetrics experts guide the treatment of cardio-obstetric patients before, during, and after pregnancy. This scientific statement aims to highlight the role of specialized cardio-obstetric anesthesiology care, presenting a systematic approach to the care of these patients from the anesthesiology perspective. The anesthesiologist is a critical part of the pregnancy heart team as the perioperative physician who is trained to prevent or promptly recognize and treat patients with peripartum cardiovascular decompensation. Maternal morbidity is attenuated with expert anesthesiology peripartum care, which includes the management of neuraxial anesthesia, inotrope and vasopressor support, transthoracic echocardiography, optimization of delivery location, and consideration of advanced critical care and mechanical support when needed. Standardizing the anesthesiology approach to patients with high peripartum cardiovascular risk and ensuring that cardio-obstetrics patients have access to the appropriate care team, facilities, and advanced cardiovascular therapies will contribute to improving peripartum morbidity and mortality.

Tailoring Hydrophobicity and Pore Environment in Physisorbents for Improved Carbon Dioxide Capture under High Humidity.

CALF-20, a Zn-triazolate-based metal-organic framework (MOF), is one of the most promising adsorbent materials for CO2 capture. However, competitive adsorption of water severely limits its performance when the relative humidity (RH) exceeds 40%, limiting the potential implementation of CALF-20 in practical settings where CO2 is saturated with moisture, such as postcombustion flue gas. In this work, three newly designed MOFs related to CALF-20, denoted as NU-220, CALF-20M-w, and CALF-20M-e that feature hydrophobic methyltriazolate linkers, are presented. Inclusion of methyl groups in the linker is proposed as a strategy to improve the uptake of CO2 in the presence of water. Notably, both CALF-20M-w and CALF-20M-e retain over 20% of their initial CO2 capture efficiency at 70% RH─a threshold at which CALF-20 shows negligible CO2 uptake. Grand canonical Monte Carlo simulations reveal that the methyl group hinders water network formation in the pores of CALF-20M-w and CALF-20M-e and enhances their CO2 selectivity over N2 in the presence of a high moisture content. Moreover, calculated radial distribution functions indicate that introducing the methyl group into the triazolate linker increases the distance between water molecules and Zn coordination bonds, offering insights into the origin of the enhanced moisture stability observed for CALF-20M-w and CALF-20M-e relative to CALF-20. Overall, this straightforward design strategy has afforded more robust sorbents that can potentially meet the challenge of effectively capturing CO2 in practical industrial applications.

Consequences of low estimated glomerular filtration rate either before or early after kidney donation.

In the general population, decreases in glomerular filtration rate (GFR) are associated with subsequent development of chronic kidney disease (CKD), cardiovascular disease (CVD), and death. It is unknown if low estimated GFR (eGFR) before or early after kidney donation was also associated with these risks. One thousand six hundred ninety-nine living donors who had both predonation and early (4-10 weeks) postdonation eGFR were included. We studied the relationships between eGFR, age at donation, and the time to sustained eGFR<45 (CKD stage 3b) and <30 mL/min/1.73m2 (CKD stage 4), hypertension, diabetes mellitus (DM), CVD, and death. Median follow-up was 12 (interquartile range, 6-21) years. Twenty-year event rates were 5.8% eGFR<45 mL/min/1.73m2; 1.2% eGFR<30 mL/min/1.73m2; 29.0% hypertension; 7.8% DM; 8.0% CVD; and 5.2% death. The median time to eGFR<45 mL/min/1.73m2 (N = 79) was 17 years, and eGFR<30 mL/min/1.73m2 (N = 22) was 25 years. Both low predonation and early postdonation eGFR were associated with eGFR<45 mL/min/1.73m2 (P < .0001) and eGFR<30 mL/min/1.73m2 (P < .006); however, the primary driver of risk for all ages was low postdonation (rather than predonation) eGFR. Predonation and postdonation eGFR were not associated with hypertension, DM, CVD, or death. Low predonation and early postdonation eGFR are risk factors for developing eGFR<45 mL/min/1.73m2 (CKD stage 3b) and <30 mL/min/1.73m2 (CKD stage 4), but not CVD, hypertension, DM, or death.

Evolution of Pancreas Transplantation At A Single Institution-50+ Years and 2,500 Transplants.

OBJECTIVE: To describe the evolution of pancreas transplantation, including improved outcomes and factors associated with improved outcomes over the past five decades. BACKGROUND: The world's first successful pancreas transplant was performed in December 1966 at the University of Minnesota. As new modalities for diabetes treatment mature, we must carefully assess the current state of pancreas transplantation to determine its ongoing role in patient care. METHODS: A single-center retrospective review of 2,500 pancreas transplants performed over >50 years in bivariate and multivariable models. Transplants were divided into six eras; outcomes are presented for the entire cohort and by era. RESULTS: All measures of patient and graft survival improved progressively through the six transplant eras. The overall death censored (DC) pancreas graft half-lives were >35 years for simultaneous pancreas and kidney (SPK), 7.1 years for pancreas after kidney (PAK), and 3.3 years for pancreas transplants alone (PTA). The 10-year DC pancreas graft survival rate in the most recent era was 86.9% for SPK recipients, 58.2% for PAK recipients, and 47.6% for PTA. Overall graft loss was most influenced by patient survival in SPK transplants, whereas graft loss in PAK and PTA recipients was more often due to graft failures. Predictors of improved pancreas graft survival were primary transplants, bladder drainage of exocrine secretions, younger donor age, and shorter preservation time. CONCLUSIONS: Pancreas outcomes have significantly improved over time via sequential, but overlapping, advances in surgical technique, immunosuppressive protocols, reduced preservation time, and the more recent reduction of immune-mediated graft loss.