Abstract: We examined whether persistence of epigenetic DNA methylation (DNA-me) alterations at specific loci over two different time points in people with diabetes are associated with metabolic memory, the prolonged beneficial effects of intensive vs. conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) Study. We compared DNA-me profiles in genomic DNA of whole blood (WB) isolated at EDIC Study baseline from 32 cases (DCCT conventional therapy group subjects showing retinopathy or albuminuria progression by EDIC Study year 10) vs. 31 controls (DCCT intensive therapy group subjects without complication progression by EDIC year 10). DNA-me was also profiled in blood monocytes (Monos) of the same patients obtained during EDIC Study years 16–17. In WB, 153 loci depicted hypomethylation, and 225 depicted hypermethylation, whereas in Monos, 155 hypomethylated loci and 247 hypermethylated loci were found (fold change ≥1.3; P 〈 0.005; cases vs. controls). Twelve annotated differentially methylated loci were common in both WB and Monos, including thioredoxin-interacting protein ( TXNIP ), known to be associated with hyperglycemia and related complications. A set of differentially methylated loci depicted similar trends of associations with prior HbA1c in both WB and Monos. In vitro, high glucose induced similar persistent hypomethylation at TXNIP in cultured THP1 Monos. These results show that DNA-me differences during the DCCT persist at certain loci associated with glycemia for several years during the EDIC Study and support an epigenetic explanation for metabolic memory.

Abstract: Cell-free DNA (cfDNA) fragmentation is nonrandom, at least partially mediated by various DNA nucleases, forming characteristic cfDNA end motifs. However, there is a paucity of tools for deciphering the relative contributions of cfDNA cleavage patterns related to underlying fragmentation factors. In this study, through non-negative matrix factorization algorithm, we used 256 5′ 4-mer end motifs to identify distinct types of cfDNA cleavage patterns, referred to as “founder” end-motif profiles (F-profiles). F-profiles were associated with different DNA nucleases based on whether such patterns were disrupted in nuclease-knockout mouse models. Contributions of individual F-profiles in a cfDNA sample could be determined by deconvolutional analysis. We analyzed 93 murine cfDNA samples of different nuclease-deficient mice and identified six types of F-profiles. F-profiles I, II, and III were linked to deoxyribonuclease 1 like 3 (DNASE1L3), deoxyribonuclease 1 (DNASE1), and DNA fragmentation factor subunit beta (DFFB), respectively. We revealed that 42.9% of plasma cfDNA molecules were attributed to DNASE1L3-mediated fragmentation, whereas 43.4% of urinary cfDNA molecules involved DNASE1-mediated fragmentation. We further demonstrated that the relative contributions of F-profiles were useful to inform pathological states, such as autoimmune disorders and cancer. Among the six F-profiles, the use of F-profile I could inform the human patients with systemic lupus erythematosus. F-profile VI could be used to detect individuals with hepatocellular carcinoma, with an area under the receiver operating characteristic curve of 0.97. F-profile VI was more prominent in patients with nasopharyngeal carcinoma undergoing chemoradiotherapy. We proposed that this profile might be related to oxidative stress.

Abstract: High-throughput sequencing technology enables population-level surveys of human genomic variation. Here, we examine the joint allele frequency distributions across continental human populations and present an approach for combining complementary aspects of whole-genome, low-coverage data and targeted high-coverage data. We apply this approach to data generated by the pilot phase of the Thousand Genomes Project, including whole-genome 2–4× coverage data for 179 samples from HapMap European, Asian, and African panels as well as high-coverage target sequencing of the exons of 800 genes from 697 individuals in seven populations. We use the site frequency spectra obtained from these data to infer demographic parameters for an Out-of-Africa model for populations of African, European, and Asian descent and to predict, by a jackknife-based approach, the amount of genetic diversity that will be discovered as sample sizes are increased. We predict that the number of discovered nonsynonymous coding variants will reach 100,000 in each population after ∼1,000 sequenced chromosomes per population, whereas ∼2,500 chromosomes will be needed for the same number of synonymous variants. Beyond this point, the number of segregating sites in the European and Asian panel populations is expected to overcome that of the African panel because of faster recent population growth. Overall, we find that the majority of human genomic variable sites are rare and exhibit little sharing among diverged populations. Our results emphasize that replication of disease association for specific rare genetic variants across diverged populations must overcome both reduced statistical power because of rarity and higher population divergence.

Abstract: Aspects of the mechanism proposed for ATL are likely to be applicable to other fusion reactions, including ER fusion mediated by the functional orthologs of ATL in yeast and plants, Sey1p, and RHD3, and the fusion of mitochondrial outer membranes by the mitofusins/Fzo1p. As in the case of ATLs, membrane fusion mediated by SNARE proteins during intracellular vesicular transport or by viral proteins often involves lipid-interacting amphipathic helices as well as a specific function for the TMs that goes beyond a role as mere membrane anchors. Our results suggest a refined model for ATL-mediated membrane fusion in which the CT and TMs of ATL cooperate with the N-terminal cytosolic domain. First, several ATL molecules in a membrane associate with each other through their TM segments ( Fig. P1 A ). Second, these complexes interact with similarly assembled ATL molecules in another membrane ( Fig. P1 B ); the interaction of ATL molecules across the two membranes requires GTP binding. It also is conceivable that the first and second steps are coordinated rather than occurring in a strictly consecutive manner. Third, GTP hydrolysis and the release of inorganic phosphate trigger a conformational change that pulls the membranes toward each other for fusion ( Fig. P1 C and D ). The nucleotide-independent oligomerization of ATL molecules might increase the efficiency of fusion by allowing several ATL molecules in each membrane to undergo the conformational changes synchronously. Local perturbation of the membrane bilayer by the CT ( Fig. P1 C ; magenta and yellow circles) also could contribute to the process by lowering the energy barrier for the approach and eventual merging of the membranes. Finally, once fusion is completed and the postfusion conformation is reached, GDP is released ( Fig. P1 D and E ), allowing the nucleotide-dependent ATL dimers to dissociate and to start a new round of fusion. Although wild-type human ATL1 can replace its functional ortholog Sey1p in Saccharomyces cerevisiae to maintain ER morphology, fusion-defective point mutants in the CT or the TMs cannot, indicating that these domains are important for fusion in vivo. The physiological relevance of the CT is supported further by the fact that C-terminal truncation mutants of human ATL1 cause HSP. Our present results show that the CT is required for efficient membrane fusion. The key feature of the CT is a conserved amphipathic helix that immediately follows the TMs. Deletion of the CT or point mutations in the helix greatly reduce the GTP-dependent fusion of ATL-containing vesicles. A synthetic peptide corresponding to the helix (CTH), but not to unrelated amphipathic helices, can act in trans to restore the fusion activity of tailless ATL. This reaction is strictly GTP dependent, as with wild-type ATL, and involves fusion of both leaflets of the bilayer and a concomitant size increase of the ATL-containing vesicles. Using biophysical assays, we demonstrated that the CTH promotes vesicle fusion by interacting directly with and perturbing the lipid bilayer. However, disturbance of the bilayer by the C-terminal helix does not cause significant lysis during fusion, as shown by an assay that measures the mixing of vesicle contents during fusion: No leakage of content was detected in the reaction with wild-type ATL, and only a low level was observed with tailless ATL in the presence of the CTH. The TM segments also play an important role in ATL-mediated membrane fusion. They do not serve as mere membrane anchors for the cytosolic domain, because they cannot be replaced by unrelated TMs. Further, point mutations in the TMs can affect ATL’s ability to catalyze fusion. Using coimmunoprecipitation experiments, we showed that the TMs mediate nucleotide-independent oligomerization of ATL molecules. Two crystal structures of the cytosolic domain of ATL ( 3 , 4 ), which likely represent pre- and postfusion conformations, suggest that ATL molecules undergo a GTP hydrolysis–induced conformational change that pulls the membranes together so that they can fuse ( Fig. P1 ). The differences in interaction surface area in the pre- and postfusion structures indicate that the energy gain from the conformational change is not large, raising the possibility that the TMs and CT, which are not included in the crystal structures, could be important for ATL-mediated fusion. Homotypic fusion, which involves the merging of identical membranes, is required for the remodeling of organelles, including the endoplasmic reticulum (ER) and mitochondria. These organelles contain membrane tubules that are connected into a network by homotypic fusion. The homotypic fusion of ER membranes is catalyzed by the atlastins (ATLs) ( 1 , 2 ), membrane-bound GTPases of the dynamin family. The physiological importance of the ATLs is indicated by the fact that mutations in one of the isoforms are known to cause a dominantly inherited form of hereditary spastic paraplegia (HSP), a neuromuscular disorder. The ATLs contain an N-terminal cytosolic domain comprising a GTPase module and a three-helix bundle, two closely spaced transmembrane (TM) segments, and a C-terminal tail (CT) ( Fig. P1 ). Here, we demonstrate that membrane fusion by ATL is achieved by the cooperation of a conformational change in the cytosolic domain with protein–lipid and protein–protein interactions within the membrane mediated by its CT and TM segments, respectively.

Abstract: De novo mutations (DNMs), or mutations that appear in an individual despite not being seen in their parents, are an important source of genetic variation whose impact is relevant to studies of human evolution, genetics, and disease. Utilizing high-coverage whole-genome sequencing data as part of the Trans-Omics for Precision Medicine (TOPMed) Program, we called 93,325 single-nucleotide DNMs across 1,465 trios from an array of diverse human populations, and used them to directly estimate and analyze DNM counts, rates, and spectra. We find a significant positive correlation between local recombination rate and local DNM rate, and that DNM rate explains a substantial portion (8.98 to 34.92%, depending on the model) of the genome-wide variation in population-level genetic variation from 41K unrelated TOPMed samples. Genome-wide heterozygosity does correlate with DNM rate, but only explains 〈 1% of variation. While we are underpowered to see small differences, we do not find significant differences in DNM rate between individuals of European, African, and Latino ancestry, nor across ancestrally distinct segments within admixed individuals. However, we did find significantly fewer DNMs in Amish individuals, even when compared with other Europeans, and even after accounting for parental age and sequencing center. Specifically, we found significant reductions in the number of C→A and T→C mutations in the Amish, which seem to underpin their overall reduction in DNMs. Finally, we calculated near-zero estimates of narrow sense heritability ( h 2 ), which suggest that variation in DNM rate is significantly shaped by nonadditive genetic effects and the environment.

Abstract: As an economic crop, pepper satisfies people’s spicy taste and has medicinal uses worldwide. To gain a better understanding of Capsicum evolution, domestication, and specialization, we present here the genome sequence of the cultivated pepper Zunla-1 ( C. annuum L.) and its wild progenitor Chiltepin ( C. annuum var. glabriusculum ). We estimate that the pepper genome expanded ∼0.3 Mya (with respect to the genome of other Solanaceae) by a rapid amplification of retrotransposons elements, resulting in a genome comprised of ∼81% repetitive sequences. Approximately 79% of 3.48-Gb scaffolds containing 34,476 protein-coding genes were anchored to chromosomes by a high-density genetic map. Comparison of cultivated and wild pepper genomes with 20 resequencing accessions revealed molecular footprints of artificial selection, providing us with a list of candidate domestication genes. We also found that dosage compensation effect of tandem duplication genes probably contributed to the pungent diversification in pepper. The Capsicum reference genome provides crucial information for the study of not only the evolution of the pepper genome but also, the Solanaceae family, and it will facilitate the establishment of more effective pepper breeding programs.

Abstract: Short-term probabilistic forecasts of the trajectory of the COVID-19 pandemic in the United States have served as a visible and important communication channel between the scientific modeling community and both the general public and decision-makers. Forecasting models provide specific, quantitative, and evaluable predictions that inform short-term decisions such as healthcare staffing needs, school closures, and allocation of medical supplies. Starting in April 2020, the US COVID-19 Forecast Hub ( https://covid19forecasthub.org/ ) collected, disseminated, and synthesized tens of millions of specific predictions from more than 90 different academic, industry, and independent research groups. A multimodel ensemble forecast that combined predictions from dozens of groups every week provided the most consistently accurate probabilistic forecasts of incident deaths due to COVID-19 at the state and national level from April 2020 through October 2021. The performance of 27 individual models that submitted complete forecasts of COVID-19 deaths consistently throughout this year showed high variability in forecast skill across time, geospatial units, and forecast horizons. Two-thirds of the models evaluated showed better accuracy than a naïve baseline model. Forecast accuracy degraded as models made predictions further into the future, with probabilistic error at a 20-wk horizon three to five times larger than when predicting at a 1-wk horizon. This project underscores the role that collaboration and active coordination between governmental public-health agencies, academic modeling teams, and industry partners can play in developing modern modeling capabilities to support local, state, and federal response to outbreaks.

Abstract: Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for “viral reconstruction” to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.

Abstract: Breast cancers are comprised of molecularly distinct subtypes that may respond differently to pathway-targeted therapies now under development. Collections of breast cancer cell lines mirror many of the molecular subtypes and pathways found in tumors, suggesting that treatment of cell lines with candidate therapeutic compounds can guide identification of associations between molecular subtypes, pathways, and drug response. In a test of 77 therapeutic compounds, nearly all drugs showed differential responses across these cell lines, and approximately one third showed subtype-, pathway-, and/or genomic aberration-specific responses. These observations suggest mechanisms of response and resistance and may inform efforts to develop molecular assays that predict clinical response.

Abstract: How promising are Mn-based compounds as potential superconductors? The schematic phase diagram ( Fig. P1 ) shows that superconductivity is achieved in compounds that are exquisitely balanced between metal and insulator and where magnetic order is on the verge of complete destruction. Achieving higher T c requires something more. Perhaps the cuprates have higher T c than the Fe-pnictides because their correlations are strong enough to make them insulating in the absence of doping. The correlations in the Mn-pnictides may be even stronger. Compounds such as LaMnPO are important because they may expose the upper bound on T c . Our calculations reveal, however, that applied pressure succeeds in delocalizing electrons in LaMnPO. The magnitude of the moments found in neutron diffraction measurements of LaMnPO is in superb agreement with the values predicted by DMFT. Given this success, we took theory as our guide. We calculated the magnetic moments and band gaps of LaMnPO at high pressures by taking as our starting point the exact crystal structures we determined from high-pressure X-ray diffraction measurements. These measurements revealed that the lattice contracts suddenly near 30 GPa (300,000 atmospheres), which corresponds to the vanishing of the ordered magnetic moment as revealed by our calculations. Moreover, we find that the band gap of LaMnPO is driven to zero between 8.5 and 16 GPa, just as we experimentally observed another lattice contraction followed by an orthorhombic distortion. While these seem like large pressures, the volume was reduced by only ∼10%, similar to the values required to induce superconductivity itself in LaFeAsO and BaFe 2 As 2 ( 3 ). Furthermore, similar distortions have been observed in Fe-based superconductors, where they are instrumental in collapsing magnetic moments and producing itinerant carriers, setting the stage for superconductivity. We first attempted to induce metallization by replacing O ions in LaMnPO with F, which has been observed to disrupt the antiferromagnetic state and lead to superconductivity in isostructural LaFeAsO ( 1 ). We observed little change in the insulating gap, magnetic ordering temperature, and ordered moment, suggesting that LaMnPO is too far from metallization to be driven there by chemical doping. We grew high-quality single crystals of one such Mn-based compound, LaMnPO, and subjected it to a number of experimental and theoretical tests to gauge the likelihood of it exhibiting superconductivity. The presence of a band gap—the defining feature of an insulator—was confirmed by optical conductivity, electrical resistivity, and photoemission measurements, and its observed magnitude was in excellent agreement with the theoretical gap obtained from our first principles calculations, establishing an important link between theory and experiment. Our calculations were carried out within dynamical mean field theory (DMFT), one of the few schemes capable of overcoming the difficulties inherent in modeling strongly correlated systems ( 2 ). Formal valence counting, in which electron transfer is estimated by considering a full valence shell for each atom, predicts divalent Mn ions in LaMnPO (La 3+ Mn 2+ P 3- O 2- ). Our DMFT calculations, which were independently confirmed by x-ray absorption measurements, show that this simple picture is not entirely correct, and substantial variations in the Mn electronic state provide the first indication that LaMnPO is close to becoming metallic, and thus nonmagnetic. Superconductivity results from mutual interactions among electrons in a material that cause their relative motions to become correlated, effectively binding them into pairs. In extreme cases, correlations can be so strong that all electrons become localized, resulting in the material becoming an insulator. Delocalized or mobile electrons are required for superconductivity, and they can be obtained from insulators either by introducing extra charges via doping or by weakening the correlations themselves. Experiments on different types of superconductors, including cuprates, indicate that superconductivity occurs just when these delocalized electrons appear. Fig. P1 summarizes the behaviors possible for different strengths of correlations and degrees of charge doping. The highest known T c ’s are found in cuprates, but lower T c ’s are found in the more weakly correlated and metallic Fe-pnictides ( 1 ). Can even higher T c ’s be found in compounds that are isostructural with Fe-pnictides but nevertheless host stronger correlations? The insulating Mn-pnictides provide an ideal testing ground for this proposal, although it is generally believed that prohibitively high pressures or large amounts of doping would be required to metallize these compounds. Fig. P1. Compounds with different strengths of correlations and degrees of charge doping. Compounds with strong correlations, such as the cuprates, are insulating until doping drives them to becoming metallic. Weakly correlated compounds such as the Fe-pnictides are always metallic, although doping destroys magnetic order. Superconductivity is nestled in the confluence of the metal-insulator transition and where magnetic order is destroyed. Mn-pnictides such as LaMnPO may be more strongly correlated than cuprates, potentially leading to larger values of T c . Superconducting materials conduct electricity without dissipating energy, but their applications have been limited by their low critical temperatures ( T c ), above which they conduct electricity as normal metals. Hopes of developing new superconductor-based technologies for energy distribution, communications, and medical imaging rely on the discovery of new materials exhibiting superconductivity above cryogenic temperatures, i.e., temperatures within about 100 degrees of absolute zero. Record high T c ’s in cuprate superconductors are achieved by chemically modifying (doping) the insulating host until it conducts electricity and loses magnetic order. Our calculations reveal that a Mn-based antiferromagnetic insulator, LaMnPO, undergoes a similar transition under the application of modest pressure, raising the possibility of high-temperature superconductivity in a new family of materials.