The human genome is generally organized into stable chromosomes, and only tumor cells are known to accumulate kilobase (kb)-sized extrachromosomal circular DNA elements (eccDNAs). However, it must be expected that kb eccDNAs exist in normal cells as a result of mutations. Here, we purify and sequence eccDNAs from muscle and blood samples from 16 healthy men, detecting ~100,000 unique eccDNA types from 16 million nuclei. Half of these structures carry genes or gene fragments and the majority are smaller than 25 kb. Transcription from eccDNAs suggests that eccDNAs reside in nuclei and recurrence of certain eccDNAs in several individuals implies DNA circularization hotspots. Gene-rich chromosomes contribute to more eccDNAs per megabase and the most transcribed protein-coding gene in muscle, TTN (titin), provides the most eccDNAs per gene. Thus, somatic genomes are rich in chromosome-derived eccDNAs that may influence phenotypes through altered gene copy numbers and transcription of full-length or truncated genes.

Mammalian circadian clocks have a hierarchical organization, governed by the suprachiasmatic nucleus (SCN) in the hypothalamus. The brain itself contains multiple loci that maintain autonomous circadian rhythmicity, but the contribution of the non-SCN clocks to this hierarchy remains unclear. We examine circadian oscillations of clock gene expression in various brain loci and discovered that in mouse, robust, higher amplitude, relatively faster oscillations occur in the choroid plexus (CP) compared to the SCN. Our computational analysis and modeling show that the CP achieves these properties by synchronization of "twist" circadian oscillators via gap-junctional connections. Using an in vitro tissue coculture model and in vivo targeted deletion of the Bmal1 gene to silence the CP circadian clock, we demonstrate that the CP clock adjusts the SCN clock likely via circulation of cerebrospinal fluid, thus finely tuning behavioral circadian rhythms.

Protein accumulation and aggregation with a concomitant loss of proteostasis often contribute to neurodegenerative diseases, and the ubiquitin-proteasome system plays a major role in protein degradation and proteostasis. Here, we show that three different proteins from Alzheimer's, Parkinson's, and Huntington's disease that misfold and oligomerize into a shared three-dimensional structure potently impair the proteasome. This study indicates that the shared conformation allows these oligomers to bind and inhibit the proteasome with low nanomolar affinity, impairing ubiquitin-dependent and ubiquitin-independent proteasome function in brain lysates. Detailed mechanistic analysis demonstrates that these oligomers inhibit the 20S proteasome through allosteric impairment of the substrate gate in the 20S core particle, preventing the 19S regulatory particle from injecting substrates into the degradation chamber. These results provide a novel molecular model for oligomer-driven impairment of proteasome function that is relevant to a variety of neurodegenerative diseases, irrespective of the specific misfolded protein that is involved.

Mechanosensitive PIEZO ion channels are evolutionarily conserved proteins whose presence is critical for normal physiology in multicellular organisms. Here we show that, in addition to mechanical stimuli, PIEZO channels are also powerfully modulated by voltage and can even switch to a purely voltage-gated mode. Mutations that cause human diseases, such as xerocytosis, profoundly shift voltage sensitivity of PIEZO1 channels toward the resting membrane potential and strongly promote voltage gating. Voltage modulation may be explained by the presence of an inactivation gate in the pore, the opening of which is promoted by outward permeation. Older invertebrate (fly) and vertebrate (fish) PIEZO proteins are also voltage sensitive, but voltage gating is a much more prominent feature of these older channels. We propose that the voltage sensitivity of PIEZO channels is a deep property co-opted to add a regulatory mechanism for PIEZO activation in widely different cellular contexts.

Regulatory T (Treg) cells contribute to the anti-inflammatory response during atherogenesis. Here we show that during atherogenesis Treg cells lose Foxp3 expression and their immunosuppressive function, leading to the conversion of a fraction of these cells into T follicular helper (Tfh) cells. We show that Tfh cells are pro-atherogenic and that their depletion reduces atherosclerosis. Mechanistically, the conversion of Treg cells to Tfh cells correlates with reduced expression of IL-2Rα and pSTAT5 levels and increased expression of IL-6Rα. In vitro, incubation of naive T cells with oxLDL prevents their differentiation into Treg cells. Furthermore, injection of lipid-free Apolipoprotein AI (ApoAI) into ApoE-/- mice reduces intracellular cholesterol levels in Treg cells and prevents their conversion into Tfh cells. Together our results suggest that ApoAI, the main protein in high-density lipoprotein particles, modulates the cellular fate of Treg cells and thus influences the immune response during atherosclerosis.

Calcific aortic valve stenosis (CAVS) is a common and life-threatening heart disease and the current treatment options cannot stop or delay its progression. A GWAS on 1009 cases and 1017 ethnically matched controls was combined with a large-scale eQTL mapping study of human aortic valve tissues (n = 233) to identify susceptibility genes for CAVS. Replication was performed in the UK Biobank, including 1391 cases and 352,195 controls. A transcriptome-wide association study (TWAS) reveals PALMD (palmdelphin) as significantly associated with CAVS. The CAVS risk alleles and increasing disease severity are both associated with decreased mRNA expression levels of PALMD in valve tissues. The top variant identified shows a similar effect and strong association with CAVS (P = 1.53 × 10-10) in UK Biobank. The identification of PALMD as a susceptibility gene for CAVS provides insights into the genetic nature of this disease, opens avenues to investigate its etiology and to develop much-needed therapeutic options.

The evolutionary basis of domestication has been a longstanding question and its genetic architecture is becoming more tractable as more domestic species become genome-enabled. Before becoming established worldwide, sheep and goats were domesticated in the fertile crescent 10,500 years before present (YBP) where their wild relatives remain. Here we sequence the genomes of wild Asiatic mouflon and Bezoar ibex in the sheep and goat domestication center and compare their genomes with that of domestics from local, traditional, and improved breeds. Among the genomic regions carrying selective sweeps differentiating domestic breeds from wild populations, which are associated among others to genes involved in nervous system, immunity and productivity traits, 20 are common to Capra and Ovis. The patterns of selection vary between species, suggesting that while common targets of selection related to domestication and improvement exist, different solutions have arisen to achieve similar phenotypic end-points within these closely related livestock species.

Prior work decoding linguistic meaning from imaging data has been largely limited to concrete nouns, using similar stimuli for training and testing, from a relatively small number of semantic categories. Here we present a new approach for building a brain decoding system in which words and sentences are represented as vectors in a semantic space constructed from massive text corpora. By efficiently sampling this space to select training stimuli shown to subjects, we maximize the ability to generalize to new meanings from limited imaging data. To validate this approach, we train the system on imaging data of individual concepts, and show it can decode semantic vector representations from imaging data of sentences about a wide variety of both concrete and abstract topics from two separate datasets. These decoded representations are sufficiently detailed to distinguish even semantically similar sentences, and to capture the similarity structure of meaning relationships between sentences.

Aortic valve stenosis (AS) is the most common valvular heart disease, and valve replacement is the only definitive treatment. Here we report a large genome-wide association (GWA) study of 2,457 Icelandic AS cases and 349,342 controls with a follow-up in up to 4,850 cases and 451,731 controls of European ancestry. We identify two new AS loci, on chromosome 1p21 near PALMD (rs7543130; odds ratio (OR) = 1.20, P = 1.2 × 10-22) and on chromosome 2q22 in TEX41 (rs1830321; OR = 1.15, P = 1.8 × 10-13). Rs7543130 also associates with bicuspid aortic valve (BAV) (OR = 1.28, P = 6.6 × 10-10) and aortic root diameter (P = 1.30 × 10-8), and rs1830321 associates with BAV (OR = 1.12, P = 5.3 × 10-3) and coronary artery disease (OR = 1.05, P = 9.3 × 10-5). The results implicate both cardiac developmental abnormalities and atherosclerosis-like processes in the pathogenesis of AS. We show that several pathways are shared by CAD and AS. Causal analysis suggests that the shared risk factors of Lp(a) and non-high-density lipoprotein cholesterol contribute substantially to the frequent co-occurence of these diseases.

BRCA1 is a tumor suppressor that regulates DNA repair by homologous recombination. Germline mutations in BRCA1 are associated with increased risk of breast and ovarian cancer and BRCA1 deficient tumors are exquisitely sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. Therefore, uncovering additional components of this DNA repair pathway is of extreme importance for further understanding cancer development and therapeutic vulnerabilities. Here, we identify EDC4, a known component of processing-bodies and regulator of mRNA decapping, as a member of the BRCA1-BRIP1-TOPBP1 complex. EDC4 plays a key role in homologous recombination by stimulating end resection at double-strand breaks. EDC4 deficiency leads to genome instability and hypersensitivity to DNA interstrand cross-linking drugs and PARP inhibitors. Lack-of-function mutations in EDC4 were detected in BRCA1/2-mutation-negative breast cancer cases, suggesting a role in breast cancer susceptibility. Collectively, this study recognizes EDC4 with a dual role in decapping and DNA repair whose inactivation phenocopies BRCA1 deficiency.

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody responses to gametocyte proteins are associated with reduced malaria transmission efficiency from humans to mosquitoes.

The brain adaptively integrates present sensory input, past experience, and options for future action. The insect mushroom body exemplifies how a central brain structure brings about such integration. Here we use a combination of systematic single-cell labeling, connectomics, transgenic silencing, and activation experiments to study the mushroom body at single-cell resolution, focusing on the behavioral architecture of its input and output neurons (MBINs and MBONs), and of the mushroom body intrinsic APL neuron. Our results reveal the identity and morphology of almost all of these 44 neurons in stage 3 Drosophila larvae. Upon an initial screen, functional analyses focusing on the mushroom body medial lobe uncover sparse and specific functions of its dopaminergic MBINs, its MBONs, and of the GABAergic APL neuron across three behavioral tasks, namely odor preference, taste preference, and associative learning between odor and taste. Our results thus provide a cellular-resolution study case of how brains organize behavior.

The intestinal immune system can respond to invading pathogens yet maintain immune tolerance to self-antigens and microbiota. Myeloid cells are central to these processes, but the signaling pathways that underlie tolerance versus inflammation are unclear. Here we show that mice lacking Calcineurin B in CD11chighMHCII+ cells (Cnb1 CD11c mice) spontaneously develop intestinal inflammation and are susceptible to induced colitis. In these mice, colitis is associated with expansion of T helper type 1 (Th1) and Th17 cell populations and a decrease in the number of FoxP3+ regulatory T (Treg) cells, and the pathology is linked to the inability of intestinal Cnb1-deficient CD11chighMHCII+ cells to express IL-2. Deleting IL-2 in CD11chighMHCII+ cells induces spontaneous colitis resembling human inflammatory bowel disease. Our findings identify that the calcineurin-NFAT-IL-2 pathway in myeloid cells is a critical regulator of intestinal homeostasis by influencing the balance of inflammatory and regulatory responses in the mouse intestine.

HIV-1 envelope (Env) mimetics are candidate components of prophylactic vaccines and potential therapeutics. Here we use a synthetic V3-glycopeptide ("Man9-V3") for structural studies of an HIV Env third variable loop (V3)-glycan directed, broadly neutralizing antibody (bnAb) lineage ("DH270"), to visualize the epitope on Env and to study how affinity maturation of the lineage proceeded. Unlike many previous V3 mimetics, Man9-V3 encompasses two key features of the V3 region recognized by V3-glycan bnAbs-the conserved GDIR motif and the N332 glycan. In our structure of an antibody fragment of a lineage member, DH270.6, in complex with the V3 glycopeptide, the conformation of the antibody-bound glycopeptide conforms closely to that of the corresponding segment in an intact HIV-1 Env trimer. An additional structure identifies roles for two critical mutations in the development of breadth. The results suggest a strategy for use of a V3 glycopeptide as a vaccine immunogen.

Despite tremendous efforts to develop stimuli-responsive enzyme delivery systems, their efficacy has been mostly limited to in vitro applications. Here we introduce, by using an approach of combining biomolecules with artificial compartments, a biomimetic strategy to create artificial organelles (AOs) as cellular implants, with endogenous stimuli-triggered enzymatic activity. AOs are produced by inserting protein gates in the membrane of polymersomes containing horseradish peroxidase enzymes selected as a model for natures own enzymes involved in the redox homoeostasis. The inserted protein gates are engineered by attaching molecular caps to genetically modified channel porins in order to induce redox-responsive control of the molecular flow through the membrane. AOs preserve their structure and are activated by intracellular glutathione levels in vitro. Importantly, our biomimetic AOs are functional in vivo in zebrafish embryos, which demonstrates the feasibility of using AOs as cellular implants in living organisms. This opens new perspectives for patient-oriented protein therapy.

The ocean's role in global climate change largely depends on its heat transport. Therefore, understanding the oceanic meridional heat transport (MHT) variability is a fundamental issue. Prevailing observational and modeling evidence suggests that MHT variability is primarily determined by the large-scale ocean circulation. Here, using new in situ observations in the eastern subpolar North Atlantic Ocean and an eddy-resolving numerical model, we show that energetic mesoscale eddies with horizontal scales of about 10-100 km profoundly modulate MHT variability on time scales from intra-seasonal to interannual. Our results reveal that the velocity changes due to mesoscale processes produce substantial variability for the MHT regionally (within sub-basins) and the subpolar North Atlantic as a whole. The findings have important implications for understanding the mechanisms for poleward heat transport variability in the subpolar North Atlantic Ocean, a key region for heat and carbon sequestration, ice-ocean interaction, and biological productivity.

In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.

The roles of RNA 5-methylcytosine (RNA:m5C) and RNA:m5C methyltransferases (RCMTs) in lineage-associated chromatin organization and drug response/resistance are unclear. Here we demonstrate that the RCMTs, namely NSUN3 and DNMT2, directly bind hnRNPK, a conserved RNA-binding protein. hnRNPK interacts with the lineage-determining transcription factors (TFs), GATA1 and SPI1/PU.1, and with CDK9/P-TEFb to recruit RNA-polymerase-II at nascent RNA, leading to formation of 5-Azacitidine (5-AZA)-sensitive chromatin structure. In contrast, NSUN1 binds BRD4 and RNA-polymerase-II to form an active chromatin structure that is insensitive to 5-AZA, but hypersensitive to the BRD4 inhibitor JQ1 and to the downregulation of NSUN1 by siRNAs. Both 5-AZA-resistant leukaemia cell lines and clinically 5-AZA-resistant myelodysplastic syndrome and acute myeloid leukaemia specimens have a significant increase in RNA:m5C and NSUN1-/BRD4-associated active chromatin. This study reveals novel RNA:m5C/RCMT-mediated chromatin structures that modulate 5-AZA response/resistance in leukaemia cells, and hence provides a new insight into treatment of leukaemia.

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.

Hyperemesis gravidarum (HG), severe nausea and vomiting of pregnancy, occurs in 0.3-2% of pregnancies and is associated with maternal and fetal morbidity. The cause of HG remains unknown, but familial aggregation and results of twin studies suggest that understanding the genetic contribution is essential for comprehending the disease etiology. Here, we conduct a genome-wide association study (GWAS) for binary (HG) and ordinal (severity of nausea and vomiting) phenotypes of pregnancy complications. Two loci, chr19p13.11 and chr4q12, are genome-wide significant (p < 5 × 10-8) in both association scans and are replicated in an independent cohort. The genes implicated at these two loci are GDF15 and IGFBP7 respectively, both known to be involved in placentation, appetite, and cachexia. While proving the casual roles of GDF15 and IGFBP7 in nausea and vomiting of pregnancy requires further study, this GWAS provides insights into the genetic risk factors contributing to the disease.